Methods for the quantitation of polypeptides

ABSTRACT

Provided are methods for quantitating an amount of a polypeptide that comprises a portion of an antibody present in a sample (e.g., a plasma or serum sample) wherein the antibody comprises a constant region (e.g., a heavy chain or light chain constant region) that comprises an engineered mutation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/246,376, filed Jan. 11, 2019, which claims the priority benefit ofU.S. Provisional Application No. 62/617,080, filed Jan. 12, 2018, thecontents of which are incorporated herein by reference in theirentirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 159792016801SEQLIST.TXT,date recorded: Jun. 1, 2021, size: 29 KB).

FIELD OF THE INVENTION

The present invention provides methods for quantitating the amount of apolypeptide that comprises an antibody constant region (or portionthereof) with an engineered mutation in a sample.

BACKGROUND OF THE INVENTION

Proteins, such as antibodies, represent a growing class of therapeutics.During pre-clinical development, candidate therapeutic proteins undergoextensive analyses in animal models to assess their pharmacokinetic(PK), pharmacodynamics (PD), and toxicokinetic (TK) characteristics, toassess their safety profiles, and to determine a safe dose forfirst-in-man studies. During clinical development, the PK, PD, and TKcharacteristics of therapeutic proteins are analyzed further in humansubjects. Data from studies in humans are used to evaluate the safetyand efficacy of the therapeutic protein, to establish dosing schedules,and/or to adjust dosages in patient subpopulations. Accordingly, it iscritical that methods for quantitating therapeutic proteins in bothpre-clinical and clinical samples be reliable and sensitive.

Ligand binding assays (LBAs) have traditionally been employed forquantitation of therapeutic proteins, due to their high sensitivity, lowcost, and high throughput. Despite these advantages, LBAs have limitedlinear dynamic range, carry a risk of cross reactivity acrossmetabolites/analogs, and are difficult to multiplex. Moreover,developing new antibodies for use in LBAs for novel therapeutic proteinsis both costly and labor-intensive.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) has emerged asa promising assay platform for quantification of therapeutic proteins.LC-MS/MS assays used for bioanalysis of therapeutic proteins inpre-clinical animal studies frequently rely upon quantification of a“surrogate peptide” (i.e., a peptide whose sequence is unique to thetherapeutic protein and absent in the proteomes of pre-clinical species)as a proxy measure of therapeutic protein. Because many therapeuticproteins are derived from human proteins, the sequence of a surrogatepeptide will likely be present in the human proteome, thus impedingaccurate quantitation of the therapeutic protein in a sample obtainedfrom a human subject.

Accordingly, there remains a need in the art for universal methods forquantification of therapeutic proteins in both pre-clinical (non-human)and clinical (human) samples. The present disclosure is directed to thisand other needs.

All references cited in this application are expressly incorporated byreference herein.

BRIEF SUMMARY OF THE INVENTION

Provided is a method for quantitating an amount of a polypeptidecomprising a portion of an antibody heavy chain constant region in asample, the method comprising: (a) digesting the sample comprising thepolypeptide comprising the portion of the antibody heavy chain constantregion, wherein the portion of the antibody heavy chain constant regioncomprises an engineered mutation, and wherein digestion produces apeptide fragment derived from the antibody heavy chain constant regionthat is between 5 and 26 amino acids long and comprises the engineeredmutation; and (b) analyzing the digested sample by mass spectrometry todetermine quantity of the peptide fragment, thereby determining thequantity of the polypeptide comprising the portion of the antibody heavychain constant region in the sample. In certain embodiments according to(or as applied to) any of the embodiments above, the peptide fragmentdoes not comprise a methionine (M) or cysteine (C). In certainembodiments according to (or as applied to) any of the embodimentsabove, the peptide fragment does not comprise an asparagine (N) followedby a glycine (G) or serine (S). In certain embodiments according to (oras applied to) any of the embodiments above, the method furthercomprises purifying and concentrating the digested sample prior to themass spectrometry analysis. In certain embodiments according to (or asapplied to) any of the embodiments above, the digested sample ispurified and concentrated via solid phase extraction (SPE). In certainembodiments according to (or as applied to) any of the embodimentsabove, the digested sample is purified and concentrated via SISCAPA(Stable Isotope Standards and Capture by Anti-Peptide Antibodies).

In certain embodiments according to (or as applied to) any of theembodiments above, the sample is a whole blood sample, a serum sample, aplasma sample, or a tissue sample. In certain embodiments according to(or as applied to) any of the embodiments above, the sample is from amouse, a non-human primate, or a human. In certain embodiments accordingto (or as applied to) any of the embodiments above, the non-humanprimate is a cynomolgus monkey or a rhesus monkey. In certainembodiments according to (or as applied to) any of the embodimentsabove, the sample is digested with at least one enzyme. In certainembodiments according to (or as applied to) any of the embodimentsabove, the at least one enzyme is trypsin, chymotrypsin, glutamylendopeptidase, lysyl endopeptidase, Asp-N, Arg-C, Glu-C, cyanogenbromide (CnBr), or combinations thereof. In certain embodimentsaccording to (or as applied to) any of the embodiments above, the massspectrometry used to determine the quantity of the polypeptide in thesample is liquid chromatography-tandem mass spectrometry analysis(LC-MS/MS).

In certain embodiments according to (or as applied to) any of theembodiments above, the polypeptide comprises a CH1 domain, and whereinthe CH1 domain comprises the engineered mutation. In certain embodimentsaccording to (or as applied to) any of the embodiments above, thepolypeptide comprises a CH2 domain, and wherein the CH2 domain comprisesthe engineered mutation. In certain embodiments according to (or asapplied to) any of the embodiments above, the polypeptide comprises aCH3 domain, and wherein the CH3 domain comprises the engineeredmutation. In certain embodiments according to (or as applied to) any ofthe embodiments above, the engineered mutation in the CH3 domain of theantibody heavy chain constant region is T366Y, T366W, T366S, L368A,T394W, T394S, F405A, F405W, Y407T, Y407V, or Y407A. In certainembodiments according to (or as applied to) any of the embodimentsabove, the engineered mutation in the CH3 domain of the antibody heavychain constant region is Y407V, and wherein the CH3 domain comprises anamino acid sequence set forth in SEQ ID NO: 6 (DGSFFLVS). In certainembodiments according to (or as applied to) any of the embodimentsabove, the digestion produces a peptide fragment comprising (such asconsisting of) the amino acid sequence TTPPVLDSDGSFFLVSK (SEQ ID NO: 7),DGSFFLVSKLTV (SEQ ID NO: 8), or GSFFLVSKLTVD (SEQ ID NO: 9). In certainembodiments according to (or as applied to) any of the embodimentsabove, the sample is digested with trypsin, and wherein the digestionproduces a peptide fragment consisting of the amino acid sequenceTTPPVLDSDGSFFLVSK (SEQ ID NO: 7). In certain embodiments according to(or as applied to) any of the embodiments above, the sample is digestedwith Asp-N, and wherein the digestion produces a peptide fragmentconsisting of the amino acid sequence DGSFFLVSKLTV (SEQ ID NO: 8). Incertain embodiments according to (or as applied to) any of theembodiments above, the sample is digested with Glu-C, and wherein thedigestion produces a peptide fragment consisting of the amino acidsequence GSFFLVSKLTVD (SEQ ID NO: 9). In certain embodiments accordingto (or as applied to) any of the embodiments above, the polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 3 or SEQ IDNO: 4. In certain embodiments according to (or as applied to) any of theembodiments above, the engineered mutation in the CH3 domain of theantibody heavy chain constant region is N434S. In certain embodimentsaccording to (or as applied to) any of the embodiments above, the sampleis digested with Glu-C and trypsin, and wherein the digestion produces apeptide fragment consisting of the amino acid sequence ALHSHYTQK (SEQ IDNO: 11). In certain embodiments according to (or as applied to) any ofthe embodiments above, the polypeptide comprises the amino acid sequenceset forth in SEQ ID NO: 2 or SEQ ID NO: 3.

In certain embodiments according to (or as applied to) any of theembodiments above, the polypeptide comprising the portion of theantibody heavy chain constant region is an antibody, an Fc-fusionprotein, or an immunoadhesin. In certain embodiments according to (or asapplied to) any of the embodiments above, the polypeptide comprising theportion of the antibody heavy chain constant region is an antibody. Incertain embodiments according to (or as applied to) any of theembodiments above, the antibody is a therapeutic antibody. In certainembodiments according to (or as applied to) any of the embodimentsabove, the antibody is a chimeric antibody, a humanized antibody, or ahuman antibody. In certain embodiments according to (or as applied to)any of the embodiments above, the antibody is a monospecific antibody, abispecific antibody, a trispecific antibody, or a multispecificantibody. In certain embodiments according to (or as applied to) any ofthe embodiments above, the antibody is a trispecific antibody. Incertain embodiments according to (or as applied to) any of theembodiments above, the trispecific antibody comprises four polypeptidechains that form three antigen binding sites that specifically bind oneor more antigen targets or target proteins, wherein a first polypeptidecomprises a structure represented by the formula: VL2-L1-VL1-L2-CL; thesecond polypeptide chain comprises a structure represented by theformula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3; the third polypeptide chaincomprises a structure represented by the formula: VH3-CH1-hinge-CH2-CH3;the fourth polypeptide chain comprises a structure represented by theformula: VL3-CL, wherein VL1 is a first immunoglobulin light chainvariable domain; VL2 is a second immunoglobulin light chain variabledomain; VL3 is a third immunoglobulin light chain variable domain; VH1is a first immunoglobulin heavy chain variable domain; VH2 is a secondimmunoglobulin heavy chain variable domain; VH3 is a thirdimmunoglobulin heavy chain variable domain; CL is an immunoglobulinlight chain constant domain; CH1 is an immunoglobulin CH1 heavy chainconstant domain; and L1, L2, L3 and L4 are amino acid linkers; whereinthe first and second polypeptides form a cross-over light chain-heavychain pair; and wherein the second polypeptide chain or the thirdpolypeptide chain comprises the amino acid sequence TTPPVLDSDGSFFLVSK(SEQ ID NO: 7), DGSFFLVSKLTV (SEQ ID NO: 8), or GSFFLVSKLTVD (SEQ ID NO:9). In certain embodiments according to (or as applied to) any of theembodiments above, the first polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 12; the second polypeptide comprisesthe amino acid sequence set forth in SEQ ID NO: 13; the thirdpolypeptide comprises the amino acid sequence set forth in SEQ ID NO:14, and the fourth polypeptide comprises the amino acid sequence setforth in SEQ ID NO: 15.

In certain embodiments according to (or as applied to) any of theembodiments above, the antibody is conjugated to a drug or a label. Incertain embodiments according to (or as applied to) any of theembodiments above, the drug is selected from a chemotherapeutic agent, acytotoxic agent, or a growth-inhibitory agent. In certain embodimentsaccording to (or as applied to) any of the embodiments above, the labelis a radioisotope, a fluorescent dye, or an enzyme. In certainembodiments according to (or as applied to) any of the embodimentsabove, the antibody is a human IgG antibody. In certain embodimentsaccording to (or as applied to) any of the embodiments above, the IgGantibody is a human IgG1 antibody or a human IgG4 antibody. In certainembodiments according to (or as applied to) any of the embodimentsabove, the antibody binds A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS,BTK, BTLA, B7DC, B7H1, B7H4/VTCN1, B7H5, B7H6, B7H7, B7RP1, B7-4, C3,C5, CCL2/MCP-1, CCL3/MIP-1a, CCL4/MIP-1b, CCL5/RANTES, CCL7/MCP-3,CCL8/mcp-2, CCL11/eotaxin, CCL15/MIP-1d, CCL17/TARC, CCL19/MIP-3b,CCL20/MIP-3a, CCL21/MIP-2, CCL24/MPIF-2/eotaxin-2, CCL25/TECK,CCL26/eotaxin-3, CCR3, CCR4, CD3, CD19, CD20, CD23/FCER2, CD24, CD27,CD28, CD38, CD39, CD40, CD70, CD80/B7-1, CD86/B7-2, CD122, CD137/41BB,CD137L, CD152/CTLA4, CD154/CD40L, CD160, CD272, CD273/PDL2, CD274/PDL1,CD275/B7H2, CD276/B7H3, CD278/ICOS, CD279/PD-1, CDH1/E-cadherin,chitinase, CLEC9, CLEC91, CRTH2, CSF-1/M-CSF, CSF-2/GM-CSF, CSF-3/GCSF,CX3CL1/SCYD1, CXCL12/SDF1, CXCL13, CXCR3, DNGR-1, ectonucleosidetriphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP,FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO,IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra,IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13,IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb/IL25, IL18, IL22, IL23, IL25, IL27,IL33, IL35, ITGB4/b4 integrin, ITK, KIR, LAGS, LAMP1, leptin, LPFS2, MHCclass II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, plateletreceptor, PROM1, S152, SISP1, SLC, SPG64, ST2/receptor for IL33, STEAP2,Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9,TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP/IL7Ra, TSLPR, TWEAK, VEGF,VISTA, Vstm3, WUCAM, or XCR/GPR5/CCXCR1. In certain embodimentsaccording to (or as applied to) any of the embodiments above, the methodis for use in pharmacokinetic study of the polypeptide comprising anantibody heavy chain constant region in a mouse, a non-human primate,and a human.

Each embodiment herein described may be combined with any otherembodiment or embodiments unless clearly indicated to the contrary. Inparticular, any feature or embodiment indicated as being preferred oradvantageous may be combined with any other feature or features orembodiment or embodiments indicated as being preferred or advantageous,unless clearly indicated to the contrary. These and other aspects of theinvention will become apparent to one of skill in the art. These andother embodiments of the invention are further described by the detaileddescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows sequence alignments of the Fc regions of several exemplarytherapeutic antibodies. Predicted tryptic peptides that comprise atleast one amino acid substitution are underlined. The cysteines presentwithin tryptic peptides are denoted with thick arrows. SEQ ID NOs: 2 and5 include the knob mutation T366W. SEQ ID NOs: 3 and 4 comprise the holemutations T366S, L368A, and Y407V.

FIGS. 2A-2C show the results of LC-MS/MS analysis of atryptically-digested trispecific construct (TRI-1) that comprises heavychains with the amino acid sequences set forth in SEQ ID NOs: 2 and 3.SEQ ID NO: 2 comprises the engineered “knob” mutation T366W, and SEQ IDNO: 3 comprises engineered “hole” mutations T366S, L368A, and Y407V.Tryptic digestion of SEQ ID NO: 3 predicted to produce a peptide havingthe amino acid sequence TTPPVLDSDGSFFLVSK (SEQ ID NO: 7), i.e., the“engineered TTPP peptide”. FIG. 2A shows the extracted ion chromatogramfor m/z 905.40-907.60. FIG. 2B shows the total ion chromatogram. FIG. 2Cshows the MS/MS spectrum of the engineered TTPP peptide.

FIGS. 3A-3D show extracted ion chromatograms of tryptically-digestedmouse serum (FIG. 3A), monkey serum (FIG. 3B), human serum (FIG. 3C),and TRI-1 in PBST (FIG. 3D). The runtime for engineered TTPP is denotedby a box.

FIGS. 4A-4D show extracted ion chromatograms of tryptically-digestedTRI-1 in mouse serum (FIG. 4A), monkey serum (FIG. 4B), human serum(FIG. 4C), and PBST (FIG. 4D). The runtime for engineered TTPP isdenoted by a box.

FIGS. 5A-5C show extracted ion chromatograms of engineered TTPP fromtryptically-digested TRI-1 in PBST, m/z=905.36-905.55. TRI-1concentrations were 20 μg/mL (FIG. 5A), 2 μg/mL (FIG. 5B), and 0.2 μg/mL(FIG. 5C). The peak areas for the TTPP peptide were 44064886 (FIG. 5A),2633166 (FIG. 5B), and 368649 (FIG. 5C).

FIGS. 6A-6C show extracted ion chromatograms of unlabeled FNWYVDGVEVHNAK(SEQ ID NO: 10) (FNWY) from tryptically-digested TRI-1 in PBST,m/z=559.90-559.98. TRI-1 concentrations were 20 μg/mL (FIG. 6A), 2 μg/mL(FIG. 6B), and 0.2 μg/mL (FIG. 6C). The peak areas for the FNWY peptidewere 70091279 (FIG. 6A), 1078300 (FIG. 6B), and 209110 (FIG. 6C).

FIGS. 7A-7C show extracted ion chromatograms of isotopically labeledFNWY (FNWY(Heavy)) from tryptically-digested SILUMAB used as an internalstandard for TRI-1 digestion in PBST, m/z=562.58-562.63. TRI-1concentrations were 20 μg/mL (FIG. 7A), 2 μg/mL (FIG. 7B), and 0.2 μg/mL(FIG. 7C). The peak areas for the FNWY peptide were 1369078 (FIG. 7A),343482 (FIG. 7B), and 473743 (FIG. 7C).

FIGS. 8A-8D show extracted ion chromatograms of engineered TTPP fromtryptically-digested TRI-1 in mouse serum, m/z=905.39-905.55. TRI-1concentrations were 0 μg/mL (control) (FIG. 8A), 0.2 μg/mL (FIG. 8B) 2μg/mL (FIG. 8C), and 20 μg/mL (FIG. 8D). The peak areas for the TTPPpeptide were ND (not detected) (FIG. 8A), ND (not detected) (FIG. 8B),2168471 (FIG. 8C), and 11833127 (FIG. 8D).

FIGS. 9A-9D show extracted ion chromatograms of unlabeled FNWY fromtryptically-digested TRI-1 in mouse serum, m/z=559.90-559.98. TRI-1concentrations were 0 μg/mL (control) (FIG. 9A), 0.2 μg/mL (FIG. 9B), 2μg/mL (FIG. 9C), and 20 μg/mL (FIG. 9D). The peak areas for the FNWYpeptide were ND (not detected) (FIG. 9A), 410330 (FIG. 9B), 2500109(FIG. 9C), and 22039621 (FIG. 9D).

FIGS. 10A-10D show extracted ion chromatograms of FNWY(Heavy) fromtryptically-digested SILUMAB used as an internal standard for TRI-1digestion in mouse serum, m/z=562.58-562.63. TRI-1 concentrations were 0μg/mL (control) (FIG. 10A), 0.2 μg/mL (FIG. 10B), 2 μg/mL (FIG. 10C),and 20 μg/mL (FIG. 10D). The peak areas for the FNWY peptide were1184181 (FIG. 10A), 2310675 (FIG. 10B), 1199642 (FIG. 10C), and 967309(FIG. 10D).

FIGS. 11A-11D show extracted ion chromatograms of engineered TTPP fromtryptically-digested TRI-1 in monkey serum, m/z=905.39-905.55. TRI-1concentrations were 0 μg/mL (control) (FIG. 11A), 0.2 μg/mL (FIG. 11B),2 μg/mL (FIG. 11C), and 20 μg/mL (FIG. 11D). The peak areas for the TTPPpeptide were ND (not detected) (FIG. 11A), ND (not detected) (FIG. 11B),1013964 (FIG. 11C), and 3751017 (FIG. 11D).

FIGS. 12A-12D show extracted ion chromatograms of unlabeled FNWY fromtryptically-digested TRI-1 in monkey serum, m/z=559.92-559.95. TRI-1concentrations were 0 μg/mL (control) (FIG. 12A), 0.2 μg/mL (FIG. 12B),2 μg/mL (FIG. 12C), and 20 μg/mL (FIG. 12D). The peak areas for the FNWYpeptide were 75675 (FIG. 12A), 97731 (FIG. 12B), 1298150 (FIG. 12C), and13378187 (FIG. 12D).

FIGS. 13A-13D show extracted ion chromatograms of FNWY (Heavy) fromtryptically-digested SILUMAB used as an internal standard for TRI-1digestion in monkey serum, m/z=562.58-562.63. TRI-1 concentrations were0 μg/mL (control) (FIG. 13A), 0.2 μg/mL (FIG. 13B), 2 μg/mL (FIG. 13C),and 20 μg/mL (FIG. 13D). The peak areas for the FNWY peptide were1446203 (FIG. 13A), 1216254 (FIG. 13B), 1271728 (FIG. 13C), and 1344983(FIG. 13D).

FIGS. 14A-14D show extracted ion chromatograms of engineered TTPP fromtryptically-digested TRI-1 in human serum, m/z=905.39-905.55. TRI-1concentrations were 0 μg/mL (control) (FIG. 14A), 0.2 μg/mL (FIG. 14B),2 μg/mL (FIG. 14C), and 20 μg/mL (FIG. 14D). The peak areas for the TTPPpeptide were ND (not detected) (FIG. 14A), ND (not detected) (FIG. 14B),194065 (FIG. 14C), and 1848332 (FIG. 14D).

FIGS. 15A-15D show comparisons of area ratio to antibody concentration.FIG. 15A shows a comparison of the area ratio of engineeredTTPP:FNWY(Heavy) versus antibody concentration of TRI-1 in PBST. FIG.15B shows a comparison of the area ratio of unlabeled FNWY:FNWY(Heavy)versus antibody concentration of TRI-1 in PBST. FIG. 15C shows acomparison of the area ratio of engineered TTPP:FNWY(Heavy) versusantibody concentration of TRI-1 in mouse serum. FIG. 15D shows acomparison of the area ratio of engineered TTPP:FNWY(Heavy) versusantibody concentration of TRI-1 in monkey serum.

FIG. 16 shows the sequences of the heavy chains and light chains of theexemplary trispecific antibody quantified in Example 2. The sequences ofthe surrogate peptides quantified in the LC-MS/MS assay are underlined.

FIG. 17 shows a schematic of the exemplary trispecific antibody andwhere the sequences of the surrogate peptides underlined in FIG. 16 arelocated in the trispecific antibody.

FIG. 18 provides an exemplary workflow diagram of a method ofquantifying the trispecific antibody using the TTPP peptide.

FIG. 19A shows an exemplary chromatogram of the TTPP peptide at LLOQ(lowest limit of quantification, 2.5 μg/ml). FIG. 19B shows an exemplarychromatogram of a stable isotope-labeled TTPP peptide (standard[¹³C11-¹⁵N2]-LTTPPVLDSDGSFFLVSK (SEQ ID NO: 20), which was used as aninternal standard in Example 3. FIG. 19C shows a calibration curve ofTTPP peptide over the range of 2.5 μg/mL-10,000 μg/mL.

FIG. 20A shows a serum calibration curve analyzed using linearregression analysis with 1/x weighting. The FIG. 20B shows a secondserum calibration curve analyzed using linear regression analysis with1/x weighting. The samples used to construct the curves in FIGS. 20A and20B were obtained from separate experiments described in Example 3.

FIG. 21A shows a comparison of relative levels of trispecific antibodyas detected by measuring levels of the TTPP peptide in serum obtainedfrom mice to which trispecific antibody was administered via LC-MS/MS.FIG. 21B shows a comparison of relative levels of trispecific antibodyas detected via ELISA in serum obtained from mice to which trispecificantibody was administered.

FIG. 22A shows the results of LC-MS/MS analysis of a blank rat serumsample. FIG. 22B shows the MS/MS spectrum of a rat serum sample spikedwith 5 μg/ml trispecific antibody.

FIG. 22C shows the results of LC-MS/MS analysis of a blank monkey serumsample. FIG. 22D shows the MS/MS spectrum of a monkey serum samplespiked with 5 μg/ml trispecific antibody.

FIG. 22E shows the results of LC-MS/MS analysis of a blank monkey serumsample. FIG. 22F shows the MS/MS spectrum of a human serum sample spikedwith 5 μg/ml trispecific antibody.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, an “antibody constant region” refers to the moreconserved region of the antibody, e.g., outside the variable domains.The term may include the light chain constant region, i.e., the CLdomain, the hinge region, as well as heavy chain constant domains CH1,CH2, CH3 and, optionally, CH4.

As used herein, the term “engineered mutation” refers to refers to amutation created by human design (i.e., the mutation did notspontaneously occur by natural causes and/or was the result ofintentional human manipulation).

As used herein, the term “antibody” may refer to intact antibodies,antibody fragments comprising at least a portion of a heavy chainconstant region (including, without limitation, Fab, F(ab′)2, Fab′-SH,Fv, scFv, or single heavy chain antibodies), provided that they exhibitthe desired biological activity; monoclonal antibodies; polyclonalantibodies; monospecific antibodies; multispecific antibodies (e.g.,bispecific antibodies and trispecific antibodies); and antibody-likeproteins.

The term “antibody” typically refers to heterotetrameric complexesincluding two light (L) chains and two heavy (H) chains. Variablenumbers of disulfide bonds connect the two heavy chains, and oneconnects each light chain to a heavy chain, in addition to intrachaindisulfide bridges. The heavy chains include a variable domain (VH)followed (N-terminus to C-terminus) by three or four constant domains.The light chains include a variable domain (VL) followed by a constantdomain (CL). Typically, mammalian light chains fall into one of twocategories based on amino acid sequence: kappa and lambda.

As used herein, the term “multispecific” when used in reference to anantibody or antibody fragment includes an antibody or antibody fragmentthat possesses two or more different binding specificities (e.g.,bispecific and trispecific antibodies). For example, each bindingspecificity may recognize a different antigen, or each bindingspecificity may recognize the same antigen with different affinityand/or precise epitope. In some embodiments, each different bindingspecificity comprises one or more different antibody antigen bindingdomains (e.g., variable domains), such that the multispecific antibodyor antibody fragment comprises, e.g., a first antigen binding domainwith a first binding specificity, a second antigen binding domain with asecond binding specificity, etc. A variety of exemplary multispecificantibody formats (e.g., bispecific and trispecific antibody formats) areknown in the art and described in further detail elsewhere herein.

Before describing the disclosed embodiments in detail, it is to beunderstood that the present disclosure is not limited to particularcompositions or biological systems, which can, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a molecule”optionally includes a combination of two or more such molecules, and thelike.

As used herein, the term “about” refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

It is understood that aspects and embodiments of the present disclosureinclude “comprising,” “consisting,” and “consisting essentially of”aspects and embodiments.

Methods for Quantitating a Polypeptide in a Sample

Mass spectrometry (MS) is a feasible alternative to ligand-bindingassays (LBAs) for the quantitative analysis of therapeutic proteins insamples obtained from, e.g., pre-clinical animal studies. The typicalprocedure for MS-based quantification entails digesting each sample togenerate peptide fragments and quantifying the amount of a specificpeptide derived from the therapeutic protein as a surrogate for thetherapeutic protein itself. Currently, “universal” surrogate peptidesderived from the constant region of a human antibody are being used ingeneric methods for the quantification of human, humanized, or chimericantibodies in nonhuman animal models. Such generic methods are based onthe principle that the sequence of the “universal” surrogate peptide isconserved throughout all human Fc domains, but is absent from species inwhich preclinical efficacy and safety studies are performed (e.g., mice,rats, dogs, monkeys, etc.). However, it is not possible to use“universal” surrogate peptides to accurately quantitate, e.g., atherapeutic protein comprising a constant region of human antibody, insamples from human patients, as the sequence of the “universal”surrogate peptide is also present in endogenous human antibodies.

Many therapeutic proteins (e.g., antibodies) that comprise a constantregion of human antibody also comprise engineered mutations that havebeen introduced into the constant region in order to, e.g., modulateeffector function, modulate serum half-life, promote heterodimerization,etc. Applicants found that peptides derived from the portion of theconstant region that contains an engineered mutation are unique in bothnon-human and human proteome backgrounds. Accordingly, the methodsdescribed herein may be used to quantitate therapeutic proteins in bothpreclinical samples (i.e., obtained from animal species) and clinicalsamples (i.e., obtained from human patients).

Provided are methods for quantitating an amount of a polypeptide in asample, wherein the polypeptide comprises a portion of an antibody heavychain constant region, and wherein the portion of the antibody heavychain constant region comprises with an engineered mutation. In someembodiments, the method comprises the steps of: (a) digesting a samplethat is suspected of comprising the polypeptide to produce a digestedsample comprising a peptide fragment that comprises the engineeredmutation; and (b) analyzing the digested sample via mass spectrometry todetermine a quantity of the peptide fragment that comprises theengineered mutation, thereby determining the quantity of thepolypeptide. Polypeptides that may be quantified using the methodsdescribed herein (e.g., fusion proteins, immunoadhesins, and antibodies)are described in further detail elsewhere herein.

In some embodiments, the methods provided herein are used inpharmacokinetic studies (such as in preclinical research or in aclinical trial). In some embodiments, the methods comprise the step ofadministering the polypeptide that comprises a portion of antibody heavychain constant region with an engineered mutation to an animal, e.g.,during pre-clinical research, prior to quantification. Exemplarynon-human animals to which the polypeptide that comprises a portion ofantibody heavy chain constant region with an engineered mutation may beadministered are described in detail below. In some embodiments, themethods comprise administering the polypeptide that comprises a portionof antibody heavy chain constant region with an engineered mutation to ahuman, e.g., a human patient during a clinical trial, prior toquantification.

In some embodiments, the sample comprising the polypeptide thatcomprises a portion of antibody heavy chain constant region with anengineered mutation is a biological sample that is derived, obtained, orseparated from an animal. In some embodiments, the animal from which thesample is derived, obtained, or separated is an animal to which thepolypeptide that comprises a portion of antibody heavy chain constantregion with an engineered mutation was administered. In someembodiments, the animal is a mammal. In some embodiments, the mammal isa human (e.g., a human patient), a non-human primate (NHP) (e.g., acynomolgus monkey, a rhesus monkey, a marmoset, a tamarin, a spidermonkey, an owl monkey, a squirrel monkey, a vervet money, a baboon, orothers), or a rodent (e.g., a mouse or rat).

In some embodiments, the sample to be analyzed according to a methodprovided herein is any sample that is suspected to contain thepolypeptide that comprises a portion of an antibody heavy chain constantregion with an engineered mutation. In some embodiments, the sample isor is derived from a bodily fluid, including, but not limited to, e.g.,blood, plasma, serum, milk, bronchial lavage, amniotic fluid, saliva,bile, or tears. In some embodiments, the sample comprises tissue orcells. In some embodiments, the sample comprises serum and a knownquantity of the peptide (i.e., “spiked” serum). In some embodiments, thespiked serum further comprises a buffer. In some embodiments, the spikedserum serves as a reference for quantifying the amount of thepolypeptide comprising a portion of an antibody heavy chain constantregion with an engineered mutation present in a sample derived,obtained, or separated from an animal.

In some embodiments, the method further comprises treating the sampleprior to digestion in order to, e.g., enrich the polypeptide thatcomprises a portion of an antibody heavy chain constant region with anengineered mutation in the sample, or to, e.g., deplete abundantproteins from a sample (such as albumin from a blood, serum, or plasmasample). In some embodiments, treating the sample comprises performingan antibody pull-down assay. Alternatively or additionally, in someembodiments, treating the sample comprises performing gelelectrophoresis, extraction, precipitation, centrifugation,chromatography (e.g., affinity capture chromatography, size exclusionchromatography, etc.), ultrafiltration, and/or one or more additionalseparation steps known to those of ordinary skill in the art.

In some embodiments, the sample is digested via chemical cleavage.Exemplary chemical reagents that cleave at specific sites inpolypeptides include, but are not limited to, e.g., formic acid,hydroxylamine, iodosobenzoic acid, and NTCB (2-nitro-5-thiocyanobenzoicacid). In some embodiments, the sample is digested with an enzyme, e.g.,an endopeptidase. In some embodiments, the enzyme is a site-specificendopeptidase. Exemplary site-specific proteases include, but are notlimited to, trypsin, chymotrypsin (high specificity, cleaves c-terminalto FYW, not before P), chymotrypsin (low specificity, cleaves c-terminalto FYWML, not before P), glutamyl endopeptidase, lysyl endopeptidase,Asp-N protease, Arg-C protease, Lys-C protease, Lys-N protease,Staphylococcus aureus V8 (also known as glutamyl endopeptidase or Glu-Cprotease), cyanogen bromide (CnBr), elastase, pepsin (pH=1.3), pepsin(pH>2), neprilysin, BNPS-skatole, caspase 1, caspase 2, caspase 3,caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9,caspase 10, clostripain, enteroinase, Factor Xa, Granzyme B,thermolysin, proline-endopeptidase, Staphylococcus peptidase I,thrombin, and Tobacco etch virus protease. In some embodiments, thesample is digested with a non-specific endopeptidase, e.g., papain orproteinase K. In some embodiments, the sample is digested with a mixturecomprising two (or more) endopeptidases (including site-specific and/ornon-site-specific). In some embodiments, the two or more endopeptidasesare used simultaneously to digest the sample. In some embodiments, thetwo or more endopeptidases are used sequentially to digest the sample.Methods for digesting polypeptides in preparation for analysis via massspectrometry are well known in the art. Exemplary methods are providedin, e.g., Gundry et al. (2009) Curr Protoc Mol Biol. doi:10.1002/0471142727.mb1025s88; Hedrick et al. (2015) Curr Protoc ChemBiol. 7(3): 201-222; Giansanti et al. (2016) Nature Protocols. 11:993-1006; Nordhoff et al. (Int J Mass Spect. 226(1): 163-180; and Zhanget al. (2014) Curr Protoc Mol Biol. doi: 10.1002/0471142727.mb1021s108.

Digestion of the sample produces a peptide fragment that comprises theengineered mutation. In some embodiments the peptide fragment producedby the digestion is between 5 and 40 amino acids in length, e.g. 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 aminoacids in length. In some embodiments, the peptide fragment does notcomprise cysteine (Cys) and/or methionine (M) residues. Alternatively oradditionally, in some embodiments, the peptide does not comprise anasparagine residue (N) followed by a glycine residue (G) or a serineresidue (S) (i.e., NG or NS). In some embodiments, the peptide fragmentdoes not comprise adjacent endopeptidase cleavage sites, e.g., two ormore site-specific endopeptidase cleavage sites separated by 6, 5, 4, 3,2, or 1 amino acids. A variety of in silico tools have been developed topredict the peptide populations that can be produced via digestion(e.g., digestion with a single enzyme or chemical cleavage reagent or adigestion with a combination of enzymes and/or chemical cleavagereagents). Such tools, which include (without limitation) PChopper,PeptideCutter, MAPPP, IPEP, MS-Digest, and Protein Digestion Simulator,are well known in the art and are publicly available, such as on theWorld Wide Web.

In some embodiments, the method further comprises purifying and/orconcentrating the digested sample prior to analysis via massspectrometry. In some embodiments, purifying and/or concentrating thedigested sample comprises performing an affinity capture chromatographyor a solid-phase extraction (SPE), and eluting a purified andconcentrated sample. (See, e.g., Gudry et al. (2009) “Preparation ofProteins and Peptides for Mass Spectrometry Analysis in a Bottom-UpProteomics Workflow.” Curr Protoc Mol Biol. CHAPTER: Unit10.25.doi:10.1002/0471142727.mb1025s88.) In some embodiments, purifying and/orconcentrating the digested sample comprises performing a Stable IsotopeStandards and Capture by Anti-Peptide Antibodies (SISCAPA). In someembodiments, the sample is subjected to an antibody pull-down assay,digested (e.g., using one or more endopeptidases known in the art), andpurified and/or concentrated via SISCAPA. Details regarding SISCAPA areprovided in, e.g., Anderson et al. (2004) “Mass spectrometricquantitation of peptides and proteins using Stable Isotope Standards andCapture by Anti-Peptide Antibodies (SISCAPA). J Proteome Res. 3(2):235-44; U.S. Pat. Nos. 7,632,686 and 9,164,089; Whiteaker et al. (2011)“Evaluation of large scale quantitative proteomic assay developmentusing peptide affinity-based mass spectrometry.” Mol Cell Proteomics.10(4):M110.005645; and Rasavi et al. (2016) “Multiplexed longitudinalmeasurement of protein biomarkers in DBS using an automated SISCAPAworkflow.” Bioanalysis. 8(15):1597-1609.

In some embodiments the digested sample is analyzed via liquidchromatography-tandem mass spectrometry (LC-MS/MS). LC-MS/MS is a methodwhere a sample mixture is first separated by liquid chromatographybefore being ionized (e.g., via electrospray ionization (ESI),atmospheric pressure chemical ionization (APCI), or atmospheric pressurephoto-ionization (APPI)) and characterized by mass-to-charge ratio andrelative abundance using two mass spectrometers in series. Detailsregarding LC-MS/MS are provided in, e.g., Grebe et al. (2011) “LC-MS/MSin the Clinical Laboratory—Where to From Here?” Clin. Biochem Review.32(1): 5-31; El-Khoury et al. “Liquid Chromatography-Tandem MassSpectrometry in the Clinical Laboratory.” J. Chrom. & Separation Tech.4:e115. doi: 10.4172/2157-7064.1000e115; Shushan et al. (2010) “A reviewof clinical diagnostic applications of liquid chromatography-tandem massspectrometry.” Mass Spec. Rev. 29:930-944, 2010.

In some embodiments, the methods provided herein can be used to detectthe peptide in a sample, wherein the concentration of the peptide in thesample is any one of about 200, 150, 100, 50, 15, 10, 5, 1, 0.5, 0.25,0.1, 0.075, 0.05, 0.025, or 0.01 ng/ml, including any range in betweenthese values.

Engineered Mutations in an Antibody Heavy Chain Constant Region

In some embodiments, polypeptide quantitated according to a methodprovided herein comprises a CH1 domain (or a portion thereof, e.g.,between 10 and 50 amino acids) that comprises an engineered mutation. Insome embodiments, the CH1 domain extends from about residue 114 to aboutresidue 223 of the antibody heavy chain, according to the Kabatnumbering system. In some embodiments, the CH1 domain extends from aboutresidue 118 to about residue 215 of the antibody heavy chain, accordingto the EU numbering system. See, e.g., International ImmunogeneticsInformation System (IMGT) Web Resources atWorldWideWeb.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html.These amino acid residue positions are based on human IgG; however, itis not intended that methods provided herein be limited to use withpolypeptides comprising the CH1 domain (or portion thereof) of a humanIgG. Corresponding CH1 domain sequences from other human Igs, andcorresponding CH1 domain sequences of other mammals (e.g., macaque,cynomolgus monkey, mouse, rat, etc.) are publicly available.

In certain embodiments, the engineered mutation in the CH1 domain (orportion thereof) comprises (such as consists of) an amino acidsubstitution, insertion, or deletion that does not affect (orsubstantially affect) the desired activity of the polypeptide comprisingthe antibody heavy chain constant region. In some embodiments, asubstitution or insertion comprises the substitution or insertion of anunnatural amino acid or a conjugated amino acid. The amino acidsubstitution, insertion, or deletion can be introduced into the CH1domain (or portion thereof) by altering the nucleic acid encoding thepolypeptide (e.g. by site-specific mutagenesis) or by peptide synthesis,as described in further detail elsewhere herein.

In certain embodiments, the polypeptide to be quantitated according to amethod provided herein comprises a human IgG1 CH1 domain (or portionthereof). In some embodiments, the engineered mutation in the humanIgG1CH1 domain (or portion thereof) drives Fc heterodimerization (e.g.,for the production of bispecific antibodies, multispecific antibodies,or one-armed antibodies). In some embodiments, the engineered mutationin the human IgG1 CH1 domain (or portion thereof) is an amino acidsubstitution at K147 or K213 (according to the EU numbering system). Insome embodiments, the engineered mutation in the human IgG1 CH1 domaincomprises (such as consists of) the amino acid substitution K147D,K147E, K213D, or K213E (according to the EU numbering system).

In certain embodiments, the polypeptide to be quantitated according to amethod provided herein comprises a CH2 domain (or a portion thereof,e.g., between 10 and 50 amino acids) that comprises an engineeredmutation. In some embodiments, the CH2 domain of a human IgG extendsfrom about residue 231 to about residue 340 of the antibody heavy chain,according to the EU numbering system. See, e.g., InternationalImmunogenetics Information System (IMGT) Web Resources atWorldWideWeb.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html.These amino acid positions are based on human IgG; however, is notintended that methods provided herein be limited to use withpolypeptides comprising the CH2 domain (or portion thereof) of a humanIgG. Corresponding CH2 domain sequences from other human Igs arepublicly available, as are the corresponding CH2 domain sequences ofother mammals (e.g., macaque, cynomolgus monkey, rat, mouse, etc.). Incertain embodiments, the engineered mutation in the CH2 domain (orportion thereof) comprises an amino acid substitution, insertion, ordeletion that does not affect (or substantially affect) the desiredactivity of the polypeptide comprising the antibody heavy chain constantregion. In some embodiments, a substitution or insertion comprises thesubstitution or insertion of an unnatural amino acid or a conjugatedamino acid.

In certain embodiments, the polypeptide to be quantitated according to amethod provided herein comprises a human IgG1 CH2 domain (or portionthereof). In some embodiments, the engineered mutation in the human IgG1CH2 domain (or portion thereof) modulates effector function. In someembodiments, the engineered mutation in the human IgG1 CH2 domain (orportion thereof) comprises an amino acid substitution at one (or more)the following residues: E233, L234, L235, G236, P238, S239, F243, T250,M252, S254, T256, P257, S267, R292, Q295, N297, S298, T299, Y300, Q311,K322, A327, L328, P329, A330, P331, 1332, and E333 (according to the EUnumbering system). In some embodiments, the engineered mutation in thehuman IgG1 CH2 domain (or portion thereof) comprises one (or more) ofthe following amino acid substitutions: E233P, L234V, L234A, L235V,L235A, G236A, P238D, S239D, F243L, T250Q, T250R, M252Y, S254T, T256E,P2571, S267E, R292P, Q295R, N297Q, N297D, N297A, S298G, S298N, S298C,S298A, S28T, T299A, Y300L, Q311I, K322A, A327G, L328E, L328F, L328W,P329G, P329N, A330S, A330L, A330V, P331S, P331V, I332E, I332Y, E333A,and E333S (according to the EU numbering system). Alternatively oradditionally, the engineered mutation in the human IgG1 CH2 domain (orportion thereof) comprises (such as consists of) ΔG236 (according to theEU numbering system).

In certain embodiments, the polypeptide to be quantitated according to amethod provided herein comprises a human IgG2 CH2 domain (or portionthereof). In some embodiments, the engineered mutation in the human IgG2CH2 domain (or portion thereof) modulates effector function. In someembodiments, the engineered mutation in the human IgG2 CH2 domain (orportion thereof) comprises an amino acid substitution at one (or more)of the following residues: K326 and E333 (according to the EU numberingsystem). In some embodiments, the engineered mutation in the human IgG1CH2 domain (or portion thereof) comprises one (or more) of the followingamino acid substitutions: K326W and E333S (according to the EU numberingsystem).

In certain embodiments, the polypeptide to be quantitated according to amethod provided herein comprises a human IgG3 CH2 domain. In someembodiments, the engineered mutation in the human IgG3 CH2 domainmodulates effector function. In some embodiments, the engineeredmutation in the human IgG3 CH2 domain comprises an amino acidsubstitution at E235 (according to the EU numbering system). In someembodiments, amino acid substitution comprises E235Y.

In certain embodiments, the polypeptide to be quantitated according to amethod provided herein comprises a human IgG4 CH2 domain (or portionthereof). In some embodiments, the engineered mutation in the human IgG4CH2 domain (or portion thereof) modulates effector function. In someembodiments, the engineered mutation in the human IgG4 CH2 domain (orportion thereof) comprises an amino acid substitution at one (or more)the following residues: S228, F234, L235, F296, G327, and P329(according to the EU numbering system). In some embodiments, amino acidsubstitution comprises one (or more) of the following amino acidsubstitutions: S228P, F234A, F234L, L235A, F296Y, G327A, P329G, andP329N.

In some embodiments, polypeptide to be quantitated according to a methodprovided herein comprises a CH3 domain (or a portion thereof, e.g.,between 10 and 50 amino acids) that comprises an engineered mutation. Insome embodiments, the CH3 domain extends from about residue 341 to aboutresidue 447, according to the EU numbering system. See, e.g.,International Immunogenetics Information System (IMGT) Web Resources atWorldWideWeb.imgt.org/IMGTScientificChart/Numbering/Hu_IGHGnber.html.These amino acid positions are based on human IgG; however, is notintended that methods provided herein be limited to use withpolypeptides comprising the CH3 domain of a human IgG. Corresponding CH3domain sequences from other human Igs are publicly available, as are thecorresponding CH3 domain sequences from other Igs of other mammals(e.g., macaque, cynomolgus monkey, rat, mouse, etc.). In certainembodiments, the engineered mutation in the CH3 domain (or portionthereof) is an amino acid substitution, insertion, or deletion that doesnot affect (or substantially affect) the desired activity of thepolypeptide comprising the antibody heavy chain constant region. In someembodiments, a substitution or insertion comprises the substitution orinsertion of an unnatural amino acid or a conjugated amino acid.

In certain embodiments, the polypeptide to be quantitated according to amethod provided herein comprises a human IgG4 CH3 domain (or portionthereof). In some embodiments, the engineered mutation in the human IgG1CH4 domain (or portion thereof) modulates effector function. In someembodiments, the engineered mutation in the human IgG1 CH3 domain (orportion thereof) comprises (such as consists of) an amino acidsubstitution at residues R409 (according to the EU numbering system). Insome embodiments, the engineered mutation in the human IgG1 CH3 domain(or portion thereof) comprises (such as consists of) the amino acidsubstitution R409K.

In certain embodiments, the polypeptide to be quantitated according to amethod provided herein comprises a human IgG1 CH3 domain (or portionthereof). In some embodiments, the engineered mutation in the human IgG1CH3 domain (or portion thereof) modulates effector function and/orimproves serum half-life. In some embodiments, the engineered mutationin the human IgG1 CH3 domain (or portion thereof) comprises an aminoacid substitution at one or more) of the following residues: N343, E380,E382, P396, M428, H433, N434, and Y436 (according to the EU numberingsystem). In some embodiments, the engineered mutation in the human IgG1CH3 domain (or portion thereof) comprises one (or more) of followingamino acid substitutions: N343A, E380A, E382V, P396L, M428I, M428L,H433K, N434S, N434F, and Y436H (according to the EU numbering system).In some embodiments, the engineered mutation in the human IgG1CH3 domain(or portion thereof) drives Fc heterodimerization (e.g., for theproduction of bispecific antibodies, multispecific antibodies, orone-armed antibodies). In some embodiments, the engineered mutation inthe human IgG1 CH3 domain (or portion thereof) comprises an amino acidsubstitution one (or more) of the following residues: Y349, 5354, R355,D356, E357, K360, T366, L368, K370, K392, T394, D399, F405, Y407, K409,and K439 (according to the EU numbering system). In some embodiments,the engineered mutation in the human IgG1 CH3 domain (or portionthereof) comprises one (or more) of the following amino acidsubstitutions: Y349C, S354C, R355D, R355E, D356K, D356R, E357K, E357R,K360D, K360E, T366R, T366K, T366N, T366Q, T366Y, T366W, T366S, T366E,T366G, L368A, L368K, L368Q, L368D, L368E, L368G, L368H, L368I, L368N,L368R, L368S, L368T, L368V, L368W, K370W, K370D, K370E, K392D, K392E,T394W, T394S, D399A, D399G, D399I, D399L, D399M, D399N, D299S, D399T,D399F, D399H, D399K, D399R, D399Y, F405A, F405W, Y407T, Y407V, Y407A,K409R, K409A, K409H, K409D, K409E, K409G, K439D, and K439E (according tothe EU numbering system).

In some embodiments, the engineered mutation in the human IgG1 CH3domain (or portion thereof) comprises (such as consists of) Y407V Insome embodiments, the polypeptide comprising an antibody heavy chainconstant region with an engineered mutation comprises an amino acidsequence set forth in SEQ ID NO: 6 (DGSFFLVS). In some embodiments,digestion of a sample comprising (or suspected of comprising) apolypeptide comprising an antibody heavy chain constant region with anengineered Y407V mutation produces a peptide fragment comprising (suchas consisting of) the amino acid sequence TTPPVLDSDGSFFLVSK (SEQ ID NO:7), DGSFFLVSKLTV (SEQ ID NO: 8), or GSFFLVSKLTVD (SEQ ID NO: 9). In someembodiments, the sample comprising (or suspected of comprising) apolypeptide comprising an antibody heavy chain constant region with anengineered Y407V mutation is digested with trypsin, and digestionproduces a peptide fragment comprising (such as consisting of) the aminoacid sequence TTPPVLDSDGSFFLVSK (SEQ ID NO: 7). In some embodiments, thesample comprising (or suspected of comprising) a polypeptide comprisingan antibody heavy chain constant region with an engineered Y407Vmutation is digested with Asp-N, and digestion produces a peptidefragment comprising (such as consisting of) the amino acid sequenceDGSFFLVSKLTV (SEQ ID NO: 8). In some embodiments, the sample comprising(or suspected of comprising) a polypeptide comprising an antibody heavychain constant region with an engineered Y407V mutation is digested withGlu-C, and digestion produces a peptide fragment comprising (such asconsisting of) the amino acid sequence GSFFLVSKLTVD (SEQ ID NO: 9).

In some embodiments, the engineered mutation in the human IgG1 CH3domain comprises (such as consists of) N434S. In some embodiments, thesample comprising (or suspected of comprising) a polypeptide comprisingan antibody heavy chain constant region with an engineered N434Smutation is digested with Glu-C and trypsin, and digestion produces apeptide fragment comprising (such as consisting of) the amino acidsequence ALHSHYTQK (SEQ ID NO: 11).

In certain embodiments, the engineered mutation is introduced into theantibody heavy chain constant region (or portion thereof) via standardmolecular biological techniques known in the art. A variety of methodsfor genetic engineering have been previously described. Such mutagenesismethods include, but are not limited to, e.g., error-prone PCR, loopshuffling, oligonucleotide-directed mutagenesis, random nucleotideinsertion or other methods prior to recombination. Further detailsregarding these methods are described in, e.g., Abou-Nadler et al.(2010) Bioengineered Bugs. 1, 337-340; Firth et al. (2005)Bioinformatics. 21, 3314-3315; Cirino et al. (2003) Methods Mol Biol.231, 3-9; Pirakitikulr (2010) Protein Sci. 19, 2336-2346; Steffens etal. (2007) J. Biomol Tech. 18, 147-149; and others.

Polypeptides Comprising a Portion of an Antibody Heavy Chain ConstantRegion

The methods provided herein can be performed using any polypeptide thatcomprises a portion of an antibody heavy chain constant region, whereinthe portion of the antibody heavy chain constant region comprises anengineered mutation. In some embodiments, the polypeptide comprises allor a portion (e.g., 10 to 50 amino acids) of a CH1 domain, CH2 domain,and/or CH3 domain, provided that the domain(s) (or portion(s) thereof)comprise an engineered mutation (e.g., an engineered mutation describedelsewhere herein). In some embodiments, the portion of the heavy chainconstant domain comprising an engineered mutation is or is derived froma mammal (e.g., a human, a non-human primate, a mouse, a rat, etc.). Insome embodiments, the portion of the heavy chain constant region thatcomprises an engineered mutation is or is derived from a human IgG (suchas an IgG1, IgG2, IgG3, or IgG4), a human IgA (such as an IgA1 or IgA2),a human IgM, a human IgE, or a human IgD. In some embodiments, theportion of the heavy chain constant region that comprises an engineeredmutation is or is derived from a mouse (such as a mouse IgA, IgD, IgE,IgG1, IgG2a, IgG2b, IgG2c, IgG3, or IgM antibody heavy chain constantregion).

In certain embodiments, the polypeptide comprising a portion of antibodyheavy chain constant region with an engineered mutation is an antibody.In some embodiments, the antibody is a human, humanized, or chimericantibody. In some embodiments the antibody comprises a constant regionof, e.g., a human IgG1, human IgG2, human IgG3, human IgG4, human IgA1,human IgA2, human IgM, human IgE, or human IgD, that comprises at leastone engineered mutation.

In certain embodiments, the polypeptide comprising a portion of antibodyheavy chain constant region with an engineered mutation is a fusionpolypeptide. In some embodiments, the fusion polypeptide is an Fc-fusionpolypeptide, which comprises the Fc domain (i.e., the CH2 and CH3domains) of an antibody heavy chain constant region. In someembodiments, the fusion polypeptide is an immunoadhesin, i.e., a fusionpolypeptide in which the functional domain of a binding protein (e.g., areceptor, ligand, or cell-adhesion polypeptide) is fused to a portion ofan antibody heavy chain constant region (typically the hinge and Fcdomain).

Antibodies

In some embodiments, the polypeptide comprising a portion of an antibodyheavy chain constant region that comprises an engineered mutation is anantibody. In some embodiments, the antibody is a heterotetramericcomplex that comprises two light (L) chains and two heavy (H) chains. Insome embodiments, the two light chains and the two heavy chains areidentical. In some embodiments, the two light chains comprise differentamino acid sequences. In some embodiments, the two heavy chains comprisedifferent amino acid sequences. In some embodiments, the antibody is afull length antibody (e.g., comprising two full-length light chains andtwo full-length heavy chains). In some embodiment, the antibody is anantibody fragment that comprises a portion of a heavy chain constantdomain that comprises an engineered mutation, e.g., a Fab, a F(ab′)2, aFab′-SH, an Fv, an scFv, or a single heavy chain antibody. In certainembodiments, the antibody is mammalian antibody (such as a humanantibody, a non-human primate antibody, a mouse antibody, a ratantibody, etc.). In some embodiments, the antibody is a monospecificantibody. In some embodiments, the antibody is a multispecific antibody.

Multispecific Antibodies

Multispecific antibodies possess binding specificities against more thanone antigen (e.g., two, three, or more than three bindingspecificities). In some embodiments, the antibody quantified using amethod provided herein is a bispecific antibody. In some embodiments, abispecific antibody comprises two different binding specificities forthe same antigen (e.g., having different binding affinity and/orspecific epitope of the same antigen). In some embodiments, a bispecificantibody comprises binding specificities for two different antigens. Insome embodiments, the bispecific antibody is a full-length or intactantibody. The methods provided herein are contemplated for use withbispecific or multispecific antibody formats known in the art. Exemplarybispecific and multispecific antibody formats include, but are notlimited to, those described below.

For example, “knobs-into-holes” is a design strategy for engineeringantibody heavy chain homodimers for heterodimerization (e.g., for theproduction of bispecific antibodies, multispecific antibodies, orone-armed antibodies). Generally, such technology involves introducing aprotuberance (“knob”) at the interface of a first polypeptide (such as afirst CH3 domain in a first antibody heavy chain) and a correspondingcavity (“hole”) in the interface of a second polypeptide (such as asecond CH3 domain in a second antibody heavy chain), such that theprotuberance can be positioned in the cavity so as to promoteheterodimer formation and hinder homodimer formation. Protuberances areconstructed by replacing small amino acid side chains from the interfaceof the first polypeptide (such as a first CH3 domain in a first antibodyheavy chain) with larger side chains (e.g. arginine, phenylalanine,tyrosine or tryptophan). Compensatory cavities of identical or similarsize to the protuberances are created in the interface of the secondpolypeptide (such as a second CH3 domain in a second antibody heavychain) by replacing large amino acid side chains with smaller ones (e.g.alanine, serine, valine, or threonine). See, e.g., U.S. Pat. Nos.5,731,168; 5,807,706; 5,821,333; 7,642,228; 7,695,936; 8,216,805; and8,679,785, the contents of each of which are incorporated by referenceherein in their entirety. Exemplary sets of knobs-into-holes mutationsinclude, but not limited to, those shown in Table 1 below:

TABLE 1 Exemplary Sets of “Knobs-into-Holes” Mutations Fc domain Y407TY407A F405A T394S T366S T394W T394S T366W S354C S354C monomer 1 L368AY407T Y407A T394S T366W T366W Y407V Fc domain T366Y T366W T394W F405WT366W T366Y T366W F405W Y349C Y349C monomer 2 F405A F405W Y407A T366ST366S L368A L368A Y407V Y407A

WO 2011/131746 describes a bispecific antibody comprising asymmetricalmutations at one of positions 366, 368, 370, 399, 405, and 407(according to EU numbering) in each CH3 domain. The mutations drivedirectional “Fab-arm” or “half-molecule” exchange between twomonospecific IgG1, IgG4 or IgG4-like antibodies upon incubation underreducing conditions.

US 2009/0232811 and Schaefer et al. (2011) PNAS USA. 108(27):11187-11192 describe Crossmab technology, i.e., a bispecific antibodyformat that involves exchanging one or more heavy chain and light chaindomains within the antigen-binding fragment (Fab) of one half of thebispecific antibody. Correct association of the light chains and theircognate heavy chains is achieved by exchange of heavy-chain andlight-chain domains within the antigen binding fragment (Fab) of onehalf of the bispecific antibody. This “crossover” retains theantigen-binding affinity but makes the two arms so different thatlight-chain mispairing can no longer occur. Exemplary Crossmab formatsinclude Crossmab^(Fab) (i.e., in which the CL and VL domains areexchanged with the CH1 and VH domains, respectively), Crossmab^(VH-VL)(i.e., in which the VL and VH domains are exchanged), andCrossmab^(CH1-VCL) (i.e., in which the CH1 and CL domains areexchanged). See also WO 2009/080251, WO 2009/080252, WO 2009/080253, andWO 2009/080254, the contents of each of which is incorporated herein byreference in their entirety.

WO 2007/147901 describes a strategy for engineering antibody heavychains for heterodimerization (e.g., for the production of bispecificantibodies, multispecific antibodies, or one-armed antibodies) thatentails introducing asymmetrical mutations in the Fc domains (i.e.,K253E, D282K, K322D into a first Fc domain and D239K, E240K, and K292Dinto a second Fc domain) and in the CH/CL domains (i.e., K96E in the CH1and E15K in the CL). Such asymmetrical mutations are reported to bothdisrupt ionic interactions that stabilize homodimerization ofhalf-antibodies and promote heterodimerization of Fc domains.

WO 2009/089004 describes heteromultimeric proteins (e.g., bispecific,multispecific, or one-armed antibodies) comprising asymmetrical pairs ofmutations in the CH3-CH3 interface that make Fc homodimerizationelectrostatically unfavorable but make Fc heterodimerizationelectrostatically favorable. See, e.g., Tables 2a and 2b in WO2009/089004).

Several antibody-like-proteins with CODV (cross-over dual variable) aredescribed in Steinmetz et al. (2016) MABS. 8(5): 867-878, WO2012/135345, WO2016/116626 and U.S. Pat. Nos. 9,181,349 9,221,917, thecontents of each of which is incorporated herein by reference in theirentirety. CODV architecture results in a circular, self-containedstructure functioning as a self-supporting truss that maintains theparental antibody affinities for both antigens. A CODV antibody-likeprotein may be (a) bivalent and/or bispecific; (b) trivalent and/ortrispecific; (c) trivalent and/or bispecific; or (d) tetravalent and/orbispecific. In one exemplary format, the polypeptide comprises twopolypeptide chains having a structure represented by the formula:V_(L1)-L₁-V_(L2)-L₂-C_(L), and two polypeptide chains have a structurerepresented by the formula: V_(H2)-L₃-V_(H1)-L₄-C_(H1)-Fc, whereinV_(L1) is a first immunoglobulin light chain variable domain; V_(L2) isa second immunoglobulin light chain variable domain; V_(H1) is a firstimmunoglobulin heavy chain variable domain; V_(H2) is a secondimmunoglobulin heavy chain variable domain; C_(L) is an immunoglobulinlight chain constant domain; C_(H1) is the immunoglobulin C_(H1) heavychain constant domain; Fc comprises an immunoglobulin hinge region andC_(H2), C_(H3) immunoglobulin heavy chain constant domains; L₁, L₂, L₃,and L₄ are amino acid linkers; and wherein the polypeptides of formula Iand the polypeptides of formula II form a cross-over light chain-heavychain pair. In another exemplary format, the polypeptide comprises twopolypeptide chains that form two antigen binding sites, wherein thefirst polypeptide has a structure represented by the formula:V_(L1)-L₁-V_(L2)-L₂-C_(L)-Fc, and the second polypeptide chain has astructure represented by the formula: V_(H2)-L₃-V_(H1)-L₄-C_(H1)-Fc,wherein: V_(L1) is a first immunoglobulin light chain variable domain;V_(L2) is a second immunoglobulin light chain variable domain; V_(H1) isa first immunoglobulin heavy chain variable domain; V_(H2) is a secondimmunoglobulin heavy chain variable domain; CL is an immunoglobulinlight chain constant domain; C_(H1) is the immunoglobulin CH1 heavychain constant domain; C_(H2) is an immunoglobulin C_(H2) heavy chainconstant domain; C_(H3) is an immunoglobulin C_(H3) heavy chain constantdomain; Fc comprises an immunoglobulin hinge region and C_(H2), C_(H3)immunoglobulin heavy chain constant domains; and L₁, L₂, L₃, and L₄ areamino acid linkers; wherein the first and second polypeptides form across-over light chain-heavy chain pair. In a third exemplary format,the polypeptide comprises three polypeptide chains that form two antigenbinding sites, wherein the first polypeptide chain has a structurerepresented by the formula: V_(L1)-L₁-V_(L2)-L₂-C_(L), the secondpolypeptide chain has a structure represented by the formula:V_(H2)-L₃-V_(H1)-L₄-C_(H1)-Fc, the third polypeptide chain comprises anantibody Fc region, wherein: V_(L1) is a first immunoglobulin lightchain variable domain; V_(L2) is a second immunoglobulin light chainvariable domain; V_(H1) is a first immunoglobulin heavy chain variabledomain; V_(H2) is a second immunoglobulin heavy chain variable domain;CL is an immunoglobulin light chain constant domain; C_(H1) is theimmunoglobulin CH1 heavy chain constant domain; C_(H2) is animmunoglobulin C_(H2) heavy chain constant domain; C_(H3) is animmunoglobulin C_(H3) heavy chain constant domain; Fc comprises animmunoglobulin hinge region and C_(H2), C_(H3) immunoglobulin heavychain constant domains; and L₁, L₂, L₃, and L₄ are amino acid linkers;wherein the first and second polypeptides form a cross-over lightchain-heavy chain pair. In a fourth exemplary format, the polypeptidecomprises a first polypeptide chain comprising a structure representedby the formula: V_(L1)-L₁-V_(L2)-L₂-C_(L) and a second polypeptide chaincomprising a structure represented by the formula:V_(H2)-L₃-V_(H1)-L₄-C_(H1) wherein: V_(L1) is a first immunoglobulinlight chain variable domain; V_(L2) is a second immunoglobulin lightchain variable domain; V_(H1) is a first immunoglobulin heavy chainvariable domain; V_(H2) is a second immunoglobulin heavy chain variabledomain; C_(L) is an immunoglobulin light chain constant domain; C_(H1)is an immunoglobulin C_(H1) heavy chain constant domain; and L₁, L₂, L₃and L₄ are amino acid linkers; wherein the polypeptide of formula I andthe polypeptide of formula II form a cross-over light chain-heavy chainpair.

A bispecific tetravalent immunoglobulin known as the tetravalentbispecific tandem immunoglobulin (TBTI) or dual variable domainimmunoglobulin (DVD-Ig) was first described in Wu et al. (2007) NatBiotechnol. 25:1290-7. Like a conventional IgG, a TBTI-DVD-Ig comprisestwo heavy chains and two light chains. However, both heavy and lightchains of a TBTI-DVD-Ig comprise an additional variable domain connectedvia a flexible, naturally occurring linker sequence at the N-termini ofthe VH and VL of an existing monoclonal antibody. Accordingly, when theheavy and the light chains combine, the resulting TBTI-DVD-Ig comprisesfour antigen recognition sites. See also U.S. Pat. Nos. 9,029,508;9,109,026; 9,035,027; 9,046,513, 8,388,965; 9,732,162; 9,738,728;European Patent No. 2573121 B1, as well as WO2012/135345, the contentsof each of which is incorporated herein by reference in their entirety.

US 2017/00320967 and WO 2017/180913, the contents of which are expresslyincorporated herein by reference in their entireties, describe a bindingprotein (such as a trivalent and/or trispecific antibody) comprisingfour polypeptide chains that form three antigen binding sites. In someembodiments, the binding protein is trivalent. In some embodiments, thebinding protein is trispecific. In one exemplary format, the firstpolypeptide chain comprises a structure represented by the formula:V_(L2)-L₁-V_(L1)-L₂-CL; the second polypeptide chain comprises astructure represented by the formula:V_(H1)-L₃-V_(H2)-L₄-C_(H1)-hinge-C_(H2)-C_(H3); the third polypeptidechain comprises a structure represented by the formula:V_(H3)-C_(H1)-hinge-C_(H2)-C_(H3); the fourth polypeptide chaincomprises a structure represented by the formula: V_(L3)-CL, whereinV_(L1) is a first immunoglobulin light chain variable domain; V_(L2) isa second immunoglobulin light chain variable domain; V_(L3) is a thirdimmunoglobulin light chain variable domain; V_(H1) is a firstimmunoglobulin heavy chain variable domain; V_(H2) is a secondimmunoglobulin heavy chain variable domain; V_(H3) is a thirdimmunoglobulin heavy chain variable domain; C_(L) is an immunoglobulinlight chain constant domain; C_(H1) is an immunoglobulin C_(H1) heavychain constant domain; and L₁, L₂, L₃ and L₄ are amino acid linkers;wherein the first and second polypeptides form a cross-over lightchain-heavy chain pair; and wherein the second polypeptide chain or thethird polypeptide chain comprises the amino acid sequenceTTPPVLDSDGSFFLVSK (SEQ ID NO: 7), DGSFFLVSKLTV (SEQ ID NO: 8), orGSFFLVSKLTVD (SEQ ID NO: 9).

In some embodiments, the linkers L₁, L₂, L₃, and L₄ range from no aminoacids (length=0) to about 100 amino acids long, or less than 100, 50,40, 30, 20, or 15 amino acids or less. The linkers can also be 10, 9, 8,7, 6, 5, 4, 3, 2, or 1 amino acids long. L₁, L₂, L₃, and L₄ in onebinding protein may all have the same amino acid sequence or may allhave different amino acid sequences.

Examples of suitable linkers include a single glycine (Gly) residue; adiglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide withfour glycine residues (Gly-Gly-Gly-Gly; SEQ ID NO: 21); a peptide withfive glycine residues (Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 22); a peptidewith six glycine residues (Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 23); apeptide with seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ IDNO: 24); a peptide with eight glycine residues(Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 25). Other combinations ofamino acid residues may be used such as the peptide Gly-Gly-Gly-Gly-Ser(SEQ ID NO: 26), the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ ID NO: 27) and the peptideGly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser (SEQ ID NO:28). Other suitable linkers include a single Ser, and Val residue; thedipeptide Arg-Thr, Gln-Pro, Ser-Ser, Thr-Lys, and Ser-Leu;Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 29), Thr-Val-Ala-Ala-Pro (SEQ ID NO:30), Gln-Pro-Lys-Ala-Ala (SEQ ID NO: 39), Gln-Arg-Ile-Glu-Gly (SEQ IDNO: 31); Ala-Ser-Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 37),Arg-Thr-Val-Ala-Ala-Pro-Ser (SEQ ID NO: 32), Gly-Gln-Pro-Lys-Ala-Ala-Pro(SEQ ID NO: 33), and His-Ile-Asp-Ser-Pro-Asn-Lys (SEQ ID NO: 34). Theexamples listed above are not intended to limit the scope of thedisclosure in any way, and linkers comprising randomly selected aminoacids selected from the group consisting of valine, leucine, isoleucine,serine, threonine, lysine, arginine, histidine, aspartate, glutamate,asparagine, glutamine, glycine, and proline have been shown to besuitable in the binding proteins.

The identity and sequence of amino acid residues in the linker may varydepending on the type of secondary structural element necessary toachieve in the linker. For example, glycine, serine, and alanine arebest for linkers having maximum flexibility. Some combination ofglycine, praline, threonine, and serine are useful if a more rigid andextended linker is necessary. Any amino acid residue may be consideredas a linker in combination with other amino acid residues to constructlarger peptide linkers as necessary depending on the desired properties.

In some embodiments, the length of L₁ is at least twice the length ofL₃. In some embodiments, the length of L₂ is at least twice the lengthof L₄. In some embodiments, the length of L₁ is at least twice thelength of L₃, and the length of L₂ is at least twice the length of L₄.In some embodiments, L₁ is 3 to 12 amino acid residues in length, L₂ is3 to 14 amino acid residues in length, L₃ is 1 to 8 amino acid residuesin length, and L₄ is 1 to 3 amino acid residues in length. In someembodiments, L₁ is 5 to 10 amino acid residues in length, L₂ is 5 to 8amino acid residues in length, L₃ is 1 to 5 amino acid residues inlength, and L₄ is 1 to 2 amino acid residues in length. In someembodiments, L₁ is 7 amino acid residues in length, L₂ is 5 amino acidresidues in length, L₃ is 1 amino acid residue in length, and L₄ is 2amino acid residues in length.

In some embodiments, L₁, L₂, L₃, and/or L₄ comprise the sequenceAsp-Lys-Thr-His-Thr (SEQ ID NO: 35). In some embodiments, L₁ comprisesthe sequence Asp-Lys-Thr-His-Thr (SEQ ID NO: 35). In some embodiments,L₃ comprises the sequence Asp-Lys-Thr-His-Thr (SEQ ID NO: 35).

In some embodiments, L₁, L₂, L₃, and/or L₄ comprise the sequenceGly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 33). In some embodiments, L₁comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 33). Insome embodiments, L₁ comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro(SEQ ID NO: 33), L₂ comprises the sequence Thr-Lys-Gly-Pro-Ser-Arg (SEQID NO: 36), L₃ comprises the sequence Ser, and L₄ comprises the sequenceArg-Thr. In some embodiments, L₃ comprises the sequenceGly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 33). In some embodiments, L₁comprises the sequence Ser, L₂ comprises the sequence Arg-Thr, L₃comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 33) andL₄ comprises the sequence Thr-Lys-Gly-Pro-Ser-Arg (SEQ ID NO: 36).

In some embodiments, the first polypeptide comprises the amino acidsequence set forth in SEQ ID NO: 12; the second polypeptide comprisesthe amino acid sequence set forth in SEQ ID NO: 13; the thirdpolypeptide comprises the amino acid sequence set forth in SEQ ID NO:14, and the fourth polypeptide comprises the amino acid sequence setforth in SEQ ID NO: 15. (SEQ ID NOs: 12, 13, 14, and 15 correspond toSEQ ID NOs: 3, 4, 1, and 2 disclosed in WO 2017/074878, respectively.)The amino acid sequences of additional exemplary trispecific antibodiesare described in WO 2017/074878, the contents of which are incorporatedherein by reference in their entirety.

The strand-exchange engineered domain (SEED) platform, designed togenerate asymmetric and bispecific antibody-like molecules, is based onexchanging structurally related sequences of immunoglobulin within theconserved CH3 domains. Alternating sequences from human IgA and IgG inthe SEED CH3 domains generate two asymmetric but complementary domains,designated AG and GA. The SEED design allows efficient generation ofAG/GA heterodimers and disfavors the formation of AG/AG and GA/GAhomodimers. See, e.g., Muda et al. (2011) Prot Engineering, Design &Selection. 24(5): 447-454 and U.S. Pat. No. 8,871,912, the contents ofwhich are incorporated herein by reference in their entirety.

Other bispecific and multispecific antibody formats are described Kleinet al., (2012) mAbs 4:6, 653-663; Spiess et al. (2015) “Alternativemolecular formats and therapeutic applications for bispecificantibodies.” Mol. Immunol. 67(2 Pt A): 95-106; Egan et al. (2017) mAbs.9(1):68-84; Liu et al. (2017) Front Immunol. 8: 38; and Weidle et al.(2013) Cancer Genomics Proteomics. 10(1): 1-18.

Target Antigens

In certain embodiments, a polypeptide to be quantified according to amethod provided herein (i.e., a polypeptide comprising a portion of anantibody heavy chain constant region that comprises an engineeredmutation) specifically binds to a target antigen. Exemplary targetantigens include, without limitation, A2AR, APRIL, ATPDase, BAFF, BAFFR,BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5,B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (alsoknown as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known asRANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11(also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also knownas TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a),CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2/eotaxin-2),CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4,CD3, CD19, CD20, CD23 (also known as FCER2, a receptor for IgE), CD24,CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86(also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152(also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273(also known as PDL2), CD274 (also known as PDL1), CD275 (also known asB7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279(also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9,CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known asGM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1),CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleosidetriphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP,FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO,IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra,IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13,IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as a receptor forIL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as b4integrin), ITK, KIR, LAGS, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1,NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152,SISP1, SLC, SPG64, ST2 (also known as a receptor for IL33), STEAP2, Sykkinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1,TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor forIL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known asGPR5/CCXCR1). In some embodiments, one or more of the above antigentargets are human antigen targets. In embodiments wherein thepolypeptide comprising an antibody heavy chain constant region thatcomprises an engineered mutation is a bispecific or multispecific targetbinding protein, the target antigens may be any two (or more) of theexemplary antigens listed above.

Conjugates

In some embodiments, a polypeptide to be quantified according to amethod provided herein (i.e., a polypeptide comprising an antibody heavychain constant region that comprises an engineered mutation) isconjugated to a drug, e.g., a cytotoxic agent, a chemotherapeutic agent,a growth inhibitory agent, a toxin (e.g., an enzymatically active toxinof bacterial, fungal, plant, or animal origin, or fragments thereof).Exemplary drugs include, without limitation, daunomycin, doxorubicin,methotrexate, vindesine, BCNU, streptozoicin, vincristine, and5-fluorouracil. Exemplary toxins include, without limitation, diphtheriaA chain, nonbinding active fragments of diphtheria toxin, exotoxin Achain (from Pseudomonas aeruginosa), ricin, geldanamycin, maytansine (orother maytansinoids), auristatins, dolastatin, calicheamicin, abrin Achain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, andPAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, tricothecenes, and CC1065. Cytotoxic agents and linkers thatconjugate the agents to an antibody are known in the art; see, e.g.,Parslow, A. C. et al. (2016) Biomedicines 4:14 and Kalim, M. et al.(2017) Drug Des. Devel. Ther. 11:2265-2276.

In some embodiments, a polypeptide to be quantified according to amethod provided herein (i.e., a polypeptide comprising an antibody heavychain constant region that comprises an engineered mutation) isconjugated to detectable moiety that is capable of producing, eitherdirectly or indirectly, a detectable signal. In certain embodiments, thedetectable label is a radionuclide. A variety of radionuclides areavailable for the production of radioconjugated polypeptides for use inclinical and research purposes. Examples include ¹³C, ¹⁵N, ¹⁷O, ¹⁹F,³²P, ⁹⁰Y, ^(99m)Tc, ¹¹¹In, ¹²³I, ¹²⁵I, ¹³¹I, ¹³¹In, ¹⁵³Sm, ¹⁸⁶Re, ¹⁸⁸Re,²¹¹At, ²¹²Bi, ²¹²Pb, and radioactive isotopes of Lu, Mn, Fe, and Gd. Incertain embodiments, the detectable label is a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase,β-galactosidase, or horseradish peroxidase.

The foregoing written description is considered to be sufficient toenable one skilled in the art to practice the invention. The followingExamples are offered for illustrative purposes only, and are notintended to limit the scope of the present invention in any way. Indeed,various modifications in addition to those shown and described hereinwill become apparent to those skilled in the art from the foregoingdescription and fall within the scope of the appended claims.

EXAMPLES Example 1: Identifying a Surrogate Peptide for Use in a GenericMass Spectrometry-Based Assay for Detecting a Polypeptide Comprising anAntibody Heavy Chain Constant Region in a Sample

The following selection criteria were applied to identify a surrogatepeptide for the detection of a therapeutic antibody in a biologicalsample via mass spectrometry:

-   -   A length between 6 and 26 amino acids;    -   No adjacent endopeptidase cleavage sites;    -   No methionine (M) or cysteine (C) in the peptide sequence;    -   No asparagine followed by glycine or serine (i.e., NG or NS) in        peptide sequence;    -   Is derived from the constant region of an antibody heavy chain;        and    -   Contains at least one engineered substitution mutation (i.e.,        excluding naturally occurring polymorphisms).

First, the Fc sequences of three exemplary therapeutic antibodies werealigned. See FIG. 1 . SEQ ID NO: 1 is a portion of an anti-Target Aantibody heavy chain. SEQ ID NO: 1 does not comprise an engineeredmutation. SEQ ID NO: 2 is a portion of a first chain (i.e., “chain 1”)of a trispecific construct (i.e., “TRI-1”). TRI-1-chain 1 comprises anantibody heavy chain that comprises S354C and T366W mutations (i.e.,“knob” mutations) and M428L/N434S mutations (which extend the in vivohalf-life of TRI-1). SEQ ID NO: 3 is a portion of a second chain (i.e.,“chain 2) chain of TR1. TRI-1-chain 2 comprises an antibody heavy chainthat comprises Y349C/T366S/368A/407 V “hole” mutations and M428L/N434Smutations. SEQ ID NO: 4 is a portion of a first chain (i.e., “chain 1”)of a second trispecific construct (i.e., “TRI-2”). TRI-2-chain 1comprises an antibody heavy chain that comprises Y349C/T366S/368A/407 Vmutations (i.e., “hole” mutations) and M428L/N434S mutations. SEQ ID NO:5 is a portion of a second chain (i.e., “chain 2) of TR2. TRI-2-chain 2comprises a heavy chain that comprises S354C/T366W “knob” mutations andM428L/N434S mutations. All amino acid positions discussed above arenumbered according to the EU numbering scheme.

Candidate peptides containing at least one engineered substitutionmutation that were predicted to result from tryptic digestion of thesequences shown in FIG. 1 are underlined. The peptide TTPPVLDSDGSFFLVSK(SEQ ID NO: 7), met all the criteria listed above.

Next, BLAST searches were performed using TTPPVLDSDGSFFLVSK (SEQ ID NO:7), i.e., the “engineered TTPP peptide,” as a query sequence againstmouse, cynomolgus monkey, and human protein databases to determinewhether such sequence is found in any native proteins in these species.The only sequences exhibiting 100% amino acid identity to the engineeredTTPP peptide were known knob-in-hole antibodies. Other sequences thatexhibited 94% amino acid identity to the engineered TTPP peptidecorresponded to human Fc domains containing Y407 substitution mutationsother than Y407V and endogenous human Fc domains.

The experiments described below were performed to assess the feasibilitywith which trypsin digestion of the trispecific construct TRI-1 gaverise to the engineered TTPP peptide. Briefly, 10 μg of TRI-1 (whichcomprises SEQ ID NOs: 2 and 3 shown in FIG. 1 ) was digested using theSMART™ Digest Kit (ThermoFisher) according to manufacturer'sinstructions. The digestion reaction was incubated at 70° C. while beingshaken at 1400 rpm for 75 minutes. The digested sample was thencentrifuged at 1000×g for 2 min, and the supernatant was collected forpurification and concentration.

The required number of wells in a SOLAμ HRP solid phase extraction (SPE)plate (ThermoFisher) were equilibrated with 200 μL of acetonitrile andthen conditioned with 200 μL of 0.1% trifluoroacetic acid (TFA) inwater. Then an additional 400 μL of 0.1% TFA in water was added to eachwell. The sample supernatants were added to the wells and allowed topass through. The wells were then washed with 500 μL of 0.1% TFA inwater and then washed 500 μL of 0.1% formic acid in water. The digestedsamples were eluted with 2×25 μL of 80% acetonitrile in water with 0.1%formic acid. The eluate was diluted with 50 μL of 0.1% formic acid inwater and vortexed briefly at 400 rpm.

50 μL of each digested sample was analyzed via liquidchromatography-tandem mass spectrometry using a Waters H-Class AcquityUPLC coupled to a Thermo QExactive mass spectrometer run in typicalpeptide mapping conditions.

As shown in FIGS. 2A-2C, trypsin digestion of TRI-1 reproducibly gaverise to the engineered TTPP peptide, and the peptide was easily detectedand fragmented. The extracted ion chromatogram for m/z of 905.40-907.60,a subset of the natural isotopic range of engineered TTPP is shown inFIG. 2A. Engineered TTPP eluted at about 19 minutes. Within the totalion chromatograph (shown in FIG. 2B), engineered TTPP was a prominentpeak. Furthermore, engineered TTPP showed good fragmentation for MS/MSanalysis (FIG. 2C).

Example 2: Detection of the TTPP Peptide in Serum

Next, a series of experiments was performed to determine whether theengineered TTPP peptide could be detected in human, monkey, and mousesera via LC-MS/MS without significant background interference. Mouse andcynomolgus monkey sera were tested, as these animals are commonly usedin pre-clinical therapeutic antibody studies.

Briefly, 100 μL of vortexed Protein G magnetic beads (Pierce) and 100 μLof PBST were added into wells in a 96-well microplate (Qiagen). Theplate was gently shaken to ensure a homogenous mixture. The 96-wellplate was placed on an Alpaqua 96S Super Magnet 96 well magnet for oneminute, followed by a Qiagen Type A 96 well magnet for one minute, andthen the solution was removed. This wash cycle was repeated three moretimes with 200 μL of PBST. After washing was complete, 140 μL of PBSTwere added into each well containing the beads. Then, 30 μL of (1) mouseserum, (2) monkey serum, (3) human serum or (4) 2 μg/mL TRI-1 was addedto the wells. 30 μL of the calibrators, QC, and samples were added intoseparate wells containing the beads, PBST, and sera. The 96-well platewas then covered and shaken for 1 hour at 600 RPM and washed four times.The calibrators, QC, and samples were eluted by adding 90 μL of 0.1% TFAinto each well and gently vortexing the plate to ensure a homogeneousmixture. The plate was placed on an Alpaqua 96S Super Magnet 96 wellmagnet for one minute, followed by a Qiagen Type A 96-well magnet forone minute. Then, the eluate was transferred into a 2.0 mL 96-well deepwell Protein LoBind plate (Eppendorf). The elution procedure was thenrepeated. The pH of the eluate was neutralized with 20 μL of 1 Mtris-HCl pH 8, bringing the total volume to 200 μL. Briefly, 200 μL ofSMART digest buffer was added to each of the eluates. The samples wereincubated at 70° C. with shaking at 400 rpm for 75 minutes and then werecentrifuged at 1000×g for 2 min to pellet the SMART digest resin.

Wells in a SOLAμ HRP SPE plate were equilibrated with 200 μL ofacetonitrile and then conditioned with 200 μL of 0.1% TFA in water. Anadditional 400 μL of 0.1% TFA in water was added to each well. Eachsample supernatant was added to a separate well and allowed to passthrough. The wells were washed with 500 μL of 0.1% TFA in water and then500 μL of 0.1% formic acid in water. The digested samples were elutedwith 2×25 μL of 80% acetonitrile in water with 0.1% formic acid. Theeluate was diluted with 50 μL of 0.1% formic acid in water and vortexedbriefly at 400 rpm.

SMART digests of sample were analyzed using a Waters H-Class AcquityUPLC and Thermo QExactive mass spectrometer under standard peptidemapping conditions.

Extracted ion chromatograms for the engineered TTPP peptide (m/z905.47²⁺) in blank serum are shown in FIGS. 3A-3C, and the extracted ionchromatogram for the engineered TTPP peptide in the TRI-1 digest (2μg/mL in PBST) is shown in FIG. 3D. The peak at 20.65 min in the TRI-1digest is not present in any of the serum backgrounds tested.

To confirm that engineered TTPP peptide could be detected in serumbackgrounds, mouse, monkey, and human serum samples were spiked with 20μg/mL TRI-1 and subject to antibody pulldown, digestion, and massspectrometry analysis as described above.

Extracted ion chromatograms for the engineered TTPP peptide (m/z905.47²⁺) are shown in FIGS. 4A-4C and TRI-1 digest in PBST is shown inFIG. 4D. Upon spiking TRI-1 into each of the serum backgrounds,engineered TTPP peptide can clearly be seen in all backgrounds.

Serial dilutions of TRI-1 into the appropriate serum background to finalconcentrations of 0.2, 2, and 20 μg/mL were prepared to determinewhether signal increased proportionally to the amounts of analyte in thesample. Aliquots of serum with no antibody spike in were used asbackground controls. Pull down, digestion, SPE cleanup, and LC-MS/MSwere performed as described above.

Extracted ion chromatograms of engineered TTPP, unlabeled FNWY, and FNWY(Heavy) derived from analysis of the three tested concentrations ofTRI-1 (20, 2, and 0.2 μg/mL) in PBST are shown in FIGS. 5A-5C, 6A-6C,and 7A-7C, respectively. Extracted ion chromatograms of engineered TTPP,unlabeled FNWY, and FNWY (Heavy) derived from analysis of the fourtested concentrations of TRI-1 (20, 2, 0.2, and 0 μg/mL) in mouse serumare shown in FIGS. 8A-8C, 9A-9C, and 10A-10C, respectively. Extractedion chromatograms of engineered TTPP, unlabeled FNWY, and FNWY (Heavy)derived from analysis of the four tested concentrations of TRI-1 (20, 2,0.2, and 0 μg/mL) in monkey serum are shown in FIGS. 11A-11C, 12A-12C,and 13A-13C, respectively. Extracted ion chromatograms of engineeredTTPP derived from analysis of the four tested concentrations of TRI-1(20, 2, 0.2, and 0 μg/mL) in human serum are shown in FIGS. 14A-14C.

The peak areas for the engineered TTPP peptide and the heavy isotopepeptide FNWYVDGVEVHNAK (SEQ ID NO: 10) were determined. See Table 2below. FNWY(Heavy) is an internal standard derived from the digestion ofa stable isotope-labeled universal monoclonal antibody (SILU™ MAB).

TABLE 2 Summary of Results of Quantification Assay FNWY Area Ratio TRI-1Engineered (heavy) FNWY (Engineered Area Ratio Concentration TTPP Peak(light-unlabeled) TTPP:FNWY (FNWY:FNWY Background (μg/mL) Peak Area AreaPeak Area (heavy)) (heavy)) PBST 20 44064886 1369078 70091279 32.2 51.22 2633166 343482 1078300 7.7 3.1 0.2 368649 473743 209110 0.8 0.4 Mouse20 11833127 967309 22039621 12.2 22.8 Serum 2 2168471 1199642 25001091.8 2.1 0.2 ND 2310675 410330 ND 0.2 Monkey 20 3751017 1344983 133781872.8 9.9 Serum 2 1013964 1271728 1298150 0.8 1.0 0.2 ND 1216254 97731 ND0.1 Human 20 1848332 NA NA Serum 2 194065 NA NA 0.2 ND NA NA

The area ratios of engineered TTPP to FNWY(Heavy) for PBST, mouse serum,and monkey serum backgrounds are also shown in Table 2 and plottedversus input TRI-1 concentration in FIGS. 15A-D. The area ratio ofengineered FNWY to FNWY(Heavy) for PBST is shown in Table 2 and plottedversus input antibody concentration in FIGS. 15A, 15C, and 15D.

The data above demonstrate that there is little to no backgroundinterference for the TTPP peptide in mouse serum, monkey serum, or humanserum, and that signal generated by the TTPP peptide correlates with theamount of peptide in a sample.

TABLE 3 Amino Acid Sequences in Examples 1 and 2 SEQ ID NO. SEQUENCE  1THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLKGKEYKCKVSNKALPAPI EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGFYPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNVFSCSVMHEAL HNHYTQKSLS LSPGK  2THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLKGKEYKCKVSNKALPAPI EKTISKAKGQ PREPQVYTLP PCRDELTKNQ VSLWCLVKGFYPSDIAVFWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNVFSCSVLHEAL HSHYTQKSLS LSPG  3THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISPTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCKVSNKALPAPI EKTISKAKGQ PREPQVCTLP PSRDELTKNQ VSLSCAVKGFYPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLVSKLTV DKSRWQQGNVFSCSVLHEAL HSHYTQKSLS LSPG  4THTCPPCPAP ELLGGPSVL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCKVSNKALPAPI EKTISKAKGQ PREPQVCTLP PSRDELTKNQ VSLSCAVKGFYPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNVFSCSVMHEAL HNHYTQKSLS LSPGK  5THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCKVSNKALPAPI EKTISKAKGQ PREPQVYTLP PCRDELTKNQ VSLWELVKGFYPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNVFSCSVMHEAL HNHYTQKSLS LSPG  6 DGSFFLVS  7 TTPPVLDSDG SFFLVSK  8DGSFFLVSKL TV  9 GSFFLVSKLT VD 10 FNWYVDGVEV HNAK 11 ALHSHYTQK

Example 3: Use of the TTPP Surrogate Peptide in a Generic MassSpectrometry-Based Assay for Detecting and Quantifying an ExemplaryTrispecific Antibody

Overview

Therapeutic antibodies are attractive tools for targeting diseases andtheir market is growing at fast pace. Ligand-binding assays have beenstandard methods for measuring therapeutic antibodies. However, in orderto provide support for their development, new bioanalytical strategiesare necessary. LC-MS/MS represents a complementary method, providingspecificity and higher reproducibility requested for Good LaboratoryPractice (GLP) studies. Moreover, LC-MS/MS overcomes some limitationsassociated with ligand-binding assays (LBAs), such as matrixinterference (e.g., components in sample that may impede accuratequantification of the therapeutic antibody) and the development ofreagents, which can be costly and time-consuming.

IgGs represent the majority of therapeutic proteins currently in theclinics. In order to reduce immunogenicity, therapeutic antibodies areengineered to comprise human antibody constant region, such as human IgGconstant regions. Developing a bioanalytical LC-MS assay to quantitatesuch therapeutic antibodies has been challenging, as it is difficult toselect suitable surrogate peptides that can be used to discriminatebetween endogenous human IgGs in a sample (e.g., a plasma, serum orblood sample) and the therapeutic IgG antibody without the help of animmune-affinity step. To overcome this issue, the constant region of anexemplary therapeutic antibody was engineered to comprise a substitutionmutation, e.g., a substitution mutation that is not naturally found inhuman antibody constant regions, or the antibody constant regions ofnon-human primates or pre-clinical animals (e.g., mice). This stratagemallows an accurate quantification of the therapeutic antibody in bothnon-human and human samples without matrix interference.

Described below is an affinity extraction-free LC-MS/MS assay for a fastand accurate quantitation of a trivalent trispecific antibody against asoluble target in plasma. By avoiding an immuno-affinity step, thismethod will allow accurate quantification of total drug (sum oftarget-bound drug plus free drug). Moreover, because the substitutionmutation is in the antibody constant region, this method can be used toquantify any therapeutic protein that comprises an antibody constantregion.

Analytical Procedure

The surrogate peptides that were selected for quantification of anexemplary trispecific antibody via LC-MS/MS are shown in FIGS. 16 and 17and in Table 4 below. These surrogate peptides were selected based ontheir linear response. Peptides whose sequences are not found in humanproteins or in the proteins of non-human primates (i.e., SEQ ID NO: 7,16, and 38) were used. For absolute quantification, the engineeredpeptide TTPPVLDSDGSFFLVSK (SEQ ID NO: 7) was used (See FIGS. 16 and 17and Table 4 below). In order to assure integrity of the analyte, theratios between the areas under the peaks of TTPPVLDSDGSFFLVSK (SEQ IDNO: 7) and the other peptides listed in Table 4 were monitored in thesamples that were quantified via LC-MS/MS. The ratios stayed constant atall time points (results not shown).

TABLE 4List of Surrogate Peptides and the MS Parameters for their detectionPeptide Mass Transitions Collision Energy (V) TTPPVLDSDGSFFLVSKPrecursor Ion m/z 905.8 → 37 (SEQ ID NO: 7) Product Ion m/z 804.5DTLMISR Precursor Ion m/z 418.2 → 14 (SEQ ID NO 18)Product Ion m/z 506.3 LVIYSGSTR Precursor Ion m/z 751.9 → 21(SEQ ID NO: 16) Product Ion m/z 1036.6 DSTYSLSSTLTLSKPrecursor Ion m/z 912.2 → 30 (SEQ ID NO: 17) Product Ion m/z 811.0ESPWTFGQGTK Precursor Ion m/z 619.3 → 34 (SEQ ID NO: 38)Product Ion m/z 738.4

The stable-isotope-labeled version of TTPPVLDSDGSFFLVSK (SEQ ID NO: 7)was used as internal standard (FIG. 19B).

A fast and robust workflow was developed for supporting pre-clinicalstudies (see FIG. 18 ). The method is based on the pellet digestionprotocol described in Ouyang et al. (2012) Bioanalysis. 4(1), 17-28.Each step in the digestion protocol was optimized to maximize therecovery of the trispecific antibody from the sample and detection ofthe surrogate peptides. (The workflow for a sample from a human subjectincluded a Protein A immunopurification step prior to the denaturationstep.)

The method used only 10 μL of a plasma sample. As a small sample volumewas sufficient, the surrogate peptide quantification method is suitablefor measuring preclinical samples from, e.g., rodents. Standarddigestion time was also optimized. The best signal intensity wasobserved when the sample was digested for two hours at 37° C. To furtherincrease the sensitivity of the quantification method, samples weresubjected to solid phase extraction (SPE). The best recovery wasobtained using OASIS MCX cartridges.

Validation for Rat Plasma

Method validation was performing in rat plasma for supporting a GLPtoxicological study. A quadratic regression model was chosen with meanregression coefficient at 0.979 over three independent calibration curvewith three replicates for run and 8 nominal concentrations (FIG. 19A).Assay variability was assessed by measuring accuracy and precision ofquality control (QC) samples at LLOQ (lowest limit of quantification),LOW, MID, and HIGH of three independent preparations. Intra/inter-runaccuracy and precision are summarized in Table 5. Matrix effect wascarried out by using six different plasma batches (see Table 6 below).Stability of the antibody in plasma was evaluated in spiked human plasmastored for 1-3 and 6 months at −80° C. and at 37° C. for 24 h. Nodegradation was observed during these intervals.

TABLE 5 Inter- and Intra-Run Accuracy and Precision of three independentsamples at LLOQ, LOW, MID, and HIGH Mean Nominal Calculated Accuracy %Intra-Run Inter-Run Concentration Concentration Difference PercentPercent (μg/ml) (μg/ml) Estimate Precision Precision LLOQ 2.60 4.00 16.214.8 2.5 (n = 18) QC LOW 8.10 7.99 5.57 5.01 7.5 (n = 18) QC MID 82710.2 4.17 9.31 750 (n = 18)  QC HIGH 8220 9.53 2.61 8.63 7,500 (n = 18) 

TABLE 6 Matrix Effect on Six Independent Batches of Rat Plasma Rat 1 Rat2 Rat 3 Rat 4 Rat 5 Rat 6 (Male) (Male) (Male) (Female) (Female)(Female) Peak Areas of TTPP Peptide (SEQ ID NO: 7) in Blank PlasmaSamples 22 34 48 84 67 50 31 14 25 61 661 11 11 28 17 148 14 56 AreaLLOQ (2.5 μg/mL) 121217 155613 130475 175238 160757 157595 132624 142140148339 163371 171581 156564 125229 128498 142467 173085 165758 1175240.637% 4.58% 5.54% 19.9% 1.74% 2.28% 1.30% 14.2% 1.68% 11.2% 10.4% 1.95%1.42% 3.44% 0.822% 4.67% 2.90% 9.21%

Additional experiments were performed to determine whether 5 μg/ml oftrispecific antibody could be detected using the surrogate TTPP peptide(SEQ ID NO: 7) when spiked into monkey plasma and human plasma. As shownin FIGS. 22A-22F, the TTPP peptide was detected in the spiked rat plasma(FIG. 22B), spiked monkey plasma (FIG. 22D), and spiked human plasma(FIG. 22F), but not in blank rat plasma (FIG. 22A), blank monkey plasma(FIG. 22C) or blank human plasma (FIG. 22E).

As discussed elsewhere herein, ligand-binding assays (LBAs) such asELISAs have traditionally been used to detect and quantify therapeuticproteins (e.g., monoclonal antibodies) in biological samples. Thequantification of a monoclonal antibody via LBA typically involves usingthe target antigen as a capture agent or a detection agent. However,LBAs may generate inaccurate results if used to detect trispecificantibody and a soluble target of the trispecific antibody. For atrispecific antibody, quantification via LBA would entail developingthree different assays (i.e., one assay per target antigen), which wouldproduce three different concentration datasets, which would not bemanageable for preclinical species or human PK exposure assessment.Moreover, it would be difficult to distinguish the amount of freeantibody vs. target-bound antibody vs. total antibody in a sample usingan LBA. In this Example, a generic LC-MS assay, which can be used inboth preclinical and clinical phase, was shown to be capable ofmeasuring a trispecific antibody against a soluble target with highaccuracy. The assay was shown to be specific, as demonstrated by theabsence of interfering signals in blank plasma samples (see FIGS.20A-20F). This assay was also shown to be reproducible, as shown inTable 5 with accuracy % difference estimate and inter-run and intra-run% precision within international regulatory acceptance criteria definedby FDA and EMA guidance on bioanalytical method validation (i.e.,accuracy % not more than ±20% and precision % not more than ±20%).

Materials and Methods for Preclinical Species

Trispecific antibody was provided as a stock solution with a nominalconcentration of 25 mg/mL in 10 mM Histidine, 8% Sucrose, 0.04%polysorbate 80, pH 6.0. Calibration standard and QCs were obtained bydiluting the stock solution in rat plasma.

Pellet digestion of rat plasma containing trispecific antibody wasoptimized from the procedure previously described in Ouyang et al.(2012) Bioanalysis. 4(1), 17-28. Briefly, 10 μl of each rat plasmasample containing the trispecific antibody was placed into a 1.5 mLLobind tubes and mixed with an equal volume of PBS buffer at pH 7.4.Plasma proteins and the therapeutic antibody in each sample wereprecipitated by adding 30 μL of methanol to the tubes. After vortexing,the samples were centrifuged for 6 minutes at 2000 rcf (relativecentrifugal force) at 5° C. The supernatants were discarded and theprotein pellets were resuspended in 40 μl of 200 mM ammonium bicarbonatebuffer (ABC). 5 μl of 1M Dithiothreitol (DTT) was added to eachresuspended pellet and incubated at 56° C. for 30 min under gentleagitation. The samples were then cooled for 10 min at room temperature.Next, 10 μl of internal standard working solution (100 μg/mL) was addedto each sample and mixed. The samples were further incubated with 10 μLof Iodoacetamide (IAA) (100 mM) in 100 mM ABC buffer for 30 min undergentle rotation at room temperature while being protected from light. 30μg/mL of trypsin and 100 μL of ABC buffer were added to each sample, andthe samples were then incubated for 2 hours under gentle rotation atroom temperature.

The digestion reactions were stopped with 30 μL 10% formic acid (FA) andcentrifuged for 10 min at 16000 rcf at 5° C. Supernatants were subjectedto solid phase extraction (SPE) with OASIS MCX elution plates using apositive pressure processor manifold. Briefly, plates were equilibratedwith 200 μL of methanol and then with 200 μL of 0.5% FA. 200 μL of eachsupernatant was applied onto the plates and washed with 200 μL 0.5% FA,200 μL of 0.5% FA in 80% MeOH solution, and 200 μL of 0.5% FA in 20%MeOH solution. Samples were finally eluted with 2×25 μL of ammoniumhydroxide buffer 5%/methanol (40:60, v/v). 10 μL of extracts werediluted within 200 μL of water/formic acid (100:0.5, v/v). 10 μL of eacheluted sample was used for LC-MS/MS analysis.

LC-MS/MS analysis was performed with a SCIEX triple Quadrupole API 5500mass spectrometer running ANALYST® software version 1.6.2. The systemwas connected to an AGILENT 1290 INFINITY liquid chromatograph system.Chromatography was performed using a X-Bridge Protein BEH C4 column; 300Å (Waters 100×2.1 mm ID, 3.5 μm particle size) at 50° and a gradient ofsolvent A (0.1% FA) and B (0.1% FA in acetonitrile). After equilibrationfor 2.5 min with solvent A, the aqueous solution was brought at 55%after 5 min with a flow rate of 600 μL/min.

MS data were acquired in positive mode with an ion spray voltage of 5500V, a source temperature of 600° C., and desolvation temperature of 60°C. CE energy was optimized for each peptide (see Table 4).

Materials and Methods for Human Samples

Affinity Purification using ASSAYMAP® BRAVO liquid handling platformfrom Agilent was performed as follows: In a 96-well plate, 90 μl PBSTand 10 μl of human plasma sample were added successively. The plate wasvortexed 5 minutes and was put in position 4 maintained at approximately20° C. on the ASSAYMAP® BRAVO deck. The 96-well plate was filled with 20μl of 1M Tris, and the reservoirs were filled with the followingsolutions in successive order: Prime and equilibrate buffer(Phosphate-buffered saline (PBST)), Cartridge Wash 1 (1 M NaCl in PBST),Cartridge Wash 2 (PBST) and elution and syringe wash buffer (1% formicacid). Next, the Affinity Purification protocol on the ASSAYMAP® BRAVOliquid handling platform was run.

Denaturation, reduction, alkylation and digestion steps are performed asfollows: The 96-well plate in position 9 was centrifuged for 30 seconds.50 μl of 0.2% RAPIGEST™ surfactant in 50 mM ammonium bicarbonate and 20μL of 100 mM DL-Dithiothreitol in 100 mM ammonium bicarbonate were addedsuccessively. Next, the plate was centrifuged 30 seconds and incubatedat approximately 56° C. for 30 minutes under gentle agitation. Sampleswere cooled down at room temperature. 20 μL of 250 mM Iodoacetamide in50 mM ammonium bicarbonate and 50 μl of ISW in 200 mM ammoniumbicarbonate @ 0.01% TWEEN® 20 were added. The plate was centrifuged for30 seconds and then incubated under light-protected conditions atapproximately 20° C. for 30 minutes under gentle agitation. 100 μL of 1μg/μL fresh Trypsin in 50 mM ammonium bicarbonate were added and thesamples were incubated at approximately 37° C. overnight. Then 20 μL of10% formic acid in water (to stop the digestion) was added.

Solid Phase Extraction using an Oasis PRIME MCX μElution plate wasperformed as follows: 200 μL of each supernatant was applied onto theplates and washed with 200 μL 0.5% FA and 200 μL of 0.5% FA in 80% MeOHsolution. Samples were finally eluted with 50 μL of ammonium hydroxidebuffer 5%/methanol (40:60, v/v) and 150 μL of water at 0.5% of formicacid were added into the eluted samples. The plate was loaded intoauto-sampler tray maintained at +10° C. and 10 μL of each sample wasinjected.

LC-MS/MS analysis was performed on the human plasma samples as describedabove for pre-clinical samples.

TABLE 7 Amino Acid Sequences in Example 3 SEQ ID NO. SEQUENCE  7TTPPVLDSDG SFFLVSK 12EVRLVESGGG LVKPGGSLRL SCSASGFDFD NAWMTWVRQP PGKGLEWVGRITGPGEGWSV DYAESVKGRF TISRDNTKNT LYLEMNNVRT EDTGYYFCARTGKYYDFWSG YPPGEEYFQD WGQGTLVIVS SDKTHTQVHL TQSGPEVRKPGTSVKVSCKA PGNTLKTYDL HWVRSVPGQG LQWMGWISHE GDKKVIVERFKAKVTIDWDR STNTAYLQLS GLTSGDTAVY YCAKGSKHRL RDYALYDDDGALNWAVDVDY LSNLEFWGQG TAVTVSSDKT HTASTKGPSV FPLAPSSKSTSGGTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSVVTVPSSSLGT QTYICNVNHK PSNTKVDKKV EPKSCDKTHT CPPCPAPELLGGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVHNAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKTISKAKGQPRE PQVYTLPPCR DELTKNQVSL WCLVKGFYPS DIAVEWESNGQPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVLHEALHSH YTQKSLSLSP G 13DFVLTQSPHS LSVTPGESAS ISCKSSHSLI HGDRNNYLAW YVQKPGRSPQLLIYLASSRA SGVPDRFSGS GSDKDFTLKI SRVETEDVGT YYCMQGRESPWTFGQGTKVD IKDKTHTASE LTQDPAVSVA LKQTVTITCR GDSLRSHYASWYQKKPGQAP VLLFYGKNNR PSGIPDRFSG SASGNRASLT ITGAQAEDEADYYCSSRDKS GSRLSVFGGG TKLTVLDKTH TRTVAAPSVF IFPPSDEQLKSGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLSSTLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC 14QVQLVQSGGQ MKKPGESMRI SCRASGYEFI DCTLNWIRLA PGKRPEWMGWLKPRGGAVNY ARPLQGRVTM TRDVYSDTAF LELRSLTVDD TAVYFCTRGKNCDYNWDFEH WGRGTPVIVS SASTKGPSVF PLAPSSKSTS GGTAALGCLVKDYFPEPVTV SWNSGALTSG VHTFPAVLQS SGLYSLSSVV TVPSSSLGTQTYICNVNHKP SNTKVDKKVE PKSCDKTHTC PPCPAPELLG GPSVFLFPPKPKDTLMISRT PEVTCVVVDV SHEDPEVKFN WYVDGVEVHN AKTKPREEQYNSTYRVVSVL TVLHQDWLNG KEYKCKVSNK ALPAPIEKTI SKAKGQPREPQVCTLPPSRD ELTKNQVSLS CAVKGFYPSD IAVEWESNGQ PENNYKTTPPVLDSDGSFFL VSKLTVDKSR WQQGNVFSCS VLHEALHSHY TQKSLSLSPG 15EIVLTQSPGT LSLSPGETAI ISCRTSQYGS LAWYQQRPGQ APRLVIYSGSTRAAGIPDRF SGSRWGPDYN LTISNLESGD FGVYYCQQYE FFGQGTKVQVDIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNALQSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC 16LVIYSGSTR 17 DSTYSLSSTL TLSK 18 DTLMISR 19 TTPPVLDSDG SFFLYSK 20LTTPPVLDSD GSFFLVSK

Example 4: Proof of Concept Studies for Using the TTPP Surrogate Peptidein a Generic Mass Spectrometry-Based Assay for Detecting and Quantifyingan Exemplary Trispecific Antibody Following Administration to Mice

Materials/Methods

The materials used in this Example were: trispecific antibody, serum(mouse), Protein G resin tips (Agilent), C18 resin tips (Agilent), SILU™MAB stable isotope-labeled universal monoclonal antibody internalstandard (Sigma), Heavy labeled engineered peptide internal standard(Thermo), 50 mM ammonium bicarbonate, 8M urea with 550 mM Tris base pH8, 1M sodium chloride in phosphate buffered saline, 125 mMdithiothreitol, 250 mM iodoacetamide, phosphate buffered saline (PBS),trypsin (Promega), LysC (Promega), water, acetic acid, trifluoroaceticacid (TFA), formic acid, acetonitrile

Calibration Curve Preparation:

A calibration curve was prepared by serially diluting the trispecificantibody in mouse serum. The concentrations tested in this proof ofconcept study were 0.012, 0.024, 0.049, 0.098, 0.195, 0.391, 0.781,1.563, 3.125, 6.25, 12.5, 25.0, 50.0, 100.0, 200.0, 400.0, and 800μg/mL. Serum without trispecific antibody was used as the zero point.

Stable Isotope-Labeled Internal Standard Preparation:

On day of use, 500 μL 0.1% formic acid was added to a 100 μg vial ofSILU™ MAB stable isotope-labeled universal monoclonal antibody (SigmaAldrich) and allowed to sit at room temperature per manufacturer'sinstructions. Antibody concentration was 0.2 μg/μL.

Heavy Labeled Engineered Peptide Internal Standard Preparation:

2.5 mL 5% acetonitrile and with 0.1% formic acid was added to thelyophilized TTPP peptide (49 nmol) to achieve a concentration of 19.6pmol/μL. On the day of use, the concentration of the TTPP peptide wasdiluted to 0.08 pmol/μL in 40% acetonitrile with 0.1% formic acid.

High Throughput Assay for Quantification of Trispecific Antibody inSerum Using an Automated Liquid Handling Platform (Agilent BRAVO™)

Affinity Purification

For each calibration curve sample or the serum sample to be tested, 10μL of each sample was added to separate well in a 96-well microplate.Next, 10 μL of additional mouse serum and 30 μL of 0.2 μg/μL stableisotope-labeled universal monoclonal antibody internal standard wasadded to each well. The 96-well plate was placed on the deck of theautomated liquid handling platform (Agilent BRAVO™), and an affinitypurification protocol that included the steps below was run:

-   -   1. Initial syringe wash    -   2. Prime (100 μL PBS at 300 μL/min)    -   3. Equilibrate (50 μL PBS at 10 μL/min)    -   4. Load Sample (20 μL at 5 μL/min)    -   5. Cup Wash 1 (25 μL deionized water)    -   6. Internal Cartridge Wash 1 (50 μL 1M NaCl in PBS at 10 μL/min)    -   7. Cup Wash 2 (25 μL deionized water)    -   8. Internal Cartridge Wash 2 (50 μL PBS at 10 μL/min)    -   9. Stringent syringe wash (50 μL 5% acetic acid)    -   10. Elute (10 μL 5% acetic acid at 5 μL/min)        Digestion:

The 96-well plate containing the eluted samples was placed on the deckof the automated liquid handling platform (Agilent BRAVO™) and adigestion protocol was performed that included the steps below:

-   -   1. Transfer 40 μL 8M Urea with 550 mM Tris pH 8 to each well.    -   2. Transfer 4.3 μL 125 mM DTT to the wells and then incubate at        40° C. for 60 minutes.    -   3. Transfer 4.7 μL 250 mM IAM to the wells and then incubate at        25° C. for 60 minutes.    -   4. Transfer 111 μL of 50 mM ammonium bicarbonate to each well.    -   5. Transfer 20 μL of 0.5 μg/μL trypsin (1:10 enzyme:substrate)        and 0.2 ug/μL rLysC (1:25 enzyme:substrate) to each well and        incubate at 37° C. for 120 minutes.    -   6. Transfer 20 μL of 10% TFA to each well.    -   7. Transfer 30 μL of 0.08 pmol/μL heavy labeled engineered        peptide (peptide internal standard) to each well.

C18 Cleanup

The plate containing the digested samples was placed on the deck of theAgilent Bravo system and the “Peptide Cleanup” protocol was runconsisting of the following steps:

-   -   1. Initial syringe wash    -   2. Prime (60% acetonitrile with 0.1% TFA)    -   3. Equilibrate (50 μL 0.1% TFA at 25 μL/min)    -   4. Load Samples (190 μL at 5 μL/min)    -   5. Cup Wash (50 μL deionized water)    -   6. Internal Cartridge Wash (50 μL 0.1% TFA at 25 μL/min)    -   7. Stringent Syringe Wash (50 μL 60% acetonitrile with 0.1% TFA)    -   8. Elute (20 μL 60% acetonitrile with 0.1% TFA at 5 μL/min)

LC-MS/MS

Each eluted sample (20 μL in 60% acetonitrile with 0.1% TFA) waspromptly diluted to 100 μL with a solution of 35% acetonitrile and 0.1%formic acid to achieve 40% acetonitrile and 0.1% formic acid. Eachsample was transferred to an individual LC-MS vial and analyzed on anLC-MS/MS system. Retention time, parent mass, transitions, and optimizedfragmentation conditions vary depending on the peptide sequence andinstrument used for analysis. The TTPP peptide (TTPPVLDSDGSFFLVSK (SEQID NO: 7)) was analyzed on a WATERS® ACQUITY UPLC® I-Class liquidchromatography system coupled to a SCIEX QTRAP® 6500 LC-MS/MS systemunder the following conditions

-   -   Column: Waters Acquity UPLC Peptide BEH C18 (2.1×150 mm, 1.7 μm)    -   Column temperature: 30° C.    -   Sample Injection Gradient: Begin gradient and hold at 5% B for 1        minute. Increase to 40% B in 17 minutes. Flow rate is 0.25        mL/min. Increase flow rate to 0.4 mL/min. and 90% B for blank        injection.    -   Blank Injection Gradient: Begin gradient and hold at 90% B for 5        minutes. Flow rate is 0.4 mL/min. Decrease flow rate to 0.25        mL/min. and 5% B and hold for 3 minutes.    -   Mobile Phases: A is water with 0.1% formic acid; B is        acetonitrile with 0.1% formic acid    -   Retention time: 12.9 minutes

TABLE 8 Mass Spectrometry Settings Collision Declustering CollisionEntrance Cell Exit Q1 Mass Q3 Mass Potential Energy Potential PotentialLight 905.5 804.4 64 39 10 16 Peptide Heavy 909.5 808.4 64 40 13 16Peptide

For this Example, the calibration curve samples and 6 serum samples wereanalyzed on 2 different days. The mice were dosed twice a week byintraperitoneal injection of trispecific antibody for a total of 7doses. The study samples analyzed by LC-MS/MS were collected atsacrifice (approximately 30 hours after the last dose) and included acontrol animal dosed with saline (i.e., Mouse 6 in FIGS. 21A and 21B)and 5 mice dosed with 30 mg/kg of the trispecific antibody (i.e., Mice1-5 in FIGS. 21A and 21B).

After LC-MS/MS, each serum calibration curve was analyzed by linearregression analysis with 1/x weighting by plotting the ratio of the TTPPpeptide to its heavy labeled peptide internal standard versus thetheoretical concentration. FIGS. 20A and 20B show the results from thisanalysis. Both serum curves demonstrated good linearity (withr-squared≥0.99) and bias (≤25%) for concentrations ranging from 0.012μg/mL to 800 μg/mL.

As shown in FIG. 21A, the serum samples showed higher levels of thetrispecific peptide in most of the mice dosed with the trispecificantibody (#1-5) and lower levels in the saline dosed mouse (#6). Thepeptide levels detected in the LC-MS/MS based assay were consistentbetween the 2 assays (FIG. 21A). These samples were also analyzed usingELISA (n=4 replicates), and the results of ELISA analysis are shown inFIG. 21B. The relative levels of trispecific antibody detected byLC-MS/MS or ELISA show a consistent trend for all samples.

While the disclosure includes various embodiments, it is understood thatvariations and modifications will occur to those skilled in the art.Therefore, it is intended that the appended claims cover all suchequivalent variations that come within the scope of the disclosure. Inaddition, the section headings used herein are for organizationalpurposes only and are not to be construed as limiting the subject matterdescribed.

All references cited in this application are expressly incorporated byreference herein.

The invention claimed is:
 1. A method for quantitating an amount of atherapeutic polypeptide comprising a portion of an antibody heavy chainconstant region in a sample comprising: (a) digesting the samplecomprising the therapeutic polypeptide comprising the portion of theantibody heavy chain constant region, wherein the portion of theantibody heavy chain constant region comprises an engineered mutation,and wherein digestion produces a peptide fragment derived from theantibody heavy chain constant region that is between 5 and 26 aminoacids long and comprises the engineered mutation, (b) analyzing thedigested sample by mass spectrometry to determine quantity of thetherapeutic peptide fragment, thereby determining the quantity of thetherapeutic polypeptide comprising the portion of the antibody heavychain constant region in the sample; wherein the therapeutic polypeptidebinds to an antigen selected from the group consisting of: A2AR, APRIL,ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4/VTCN1,B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2/MCP-1, CCL3/MIP-1a,CCL4/MIP-1b, CCL5/RANTES, CCL7/MCP-3, CCL8/mcp-2, CCL11/eotaxin,CCL15/MIP-1d, CCL17/TARC, CCL19/MIP-3b, CCL20/MIP-3a, CCL21/MIP-2,CCL24/MPIF-2/eotaxin-2, CCL25/TECK, CCL26/eotaxin-3, CCR3, CCR4, CD3,CD19, CD20, CD23/FCER2, CD24, CD27, CD28, CD38, CD39, CD40, CD70,CD80/B7-1, CD86/B7-2, CD122, CD137/41BB, CD137L, CD152/CTLA4,CD154/CD40L, CD160, CD272, CD273/PDL2, CD274/PDL1, CD275/B7H2,CD276/B7H3, CD278/ICOS, CD279/PD-1, CDH1/E-cadherin, chitinase, CLEC9,CLEC91, CRTH2, CSF-1/M-CSF, CSF-2/GM-CSF, CSF-3/GCSF, CX3CL1/SCYD1,CXCL12/SDF1, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphatediphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24,GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE,IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R,IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1,IL13Ra2, IL15, IL17, IL17Rb/IL25, IL18, IL22, IL23, IL25, IL27, IL33,IL35, ITGB4/b4 integrin, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC classII, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor,PROM1, S152, SISP1, SLC, SPG64, ST2/receptor for IL33, STEAP2, Sykkinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1,TNFa, TNFRSF7, Tp55, TREM1, TSLP/IL7Ra, TSLPR, TWEAK, VEGF, VISTA,Vstm3, WUCAM, or XCR/GPR5/CCXCR1.
 2. The method of claim 1, wherein thepeptide fragment does not comprise a methionine (M), a cysteine (C), oran asparagine (N) followed by a glycine (G) or serine (S).
 3. The methodof claim 1, wherein the sample is a whole blood sample, a serum sample,a plasma sample, or a tissue sample.
 4. The method of claim 1, whereinthe sample is from a mouse, a non-human primate, or a human.
 5. Themethod of claim 4, wherein the non-human primate is a cynomolgus monkeyor a rhesus monkey.
 6. The method of claim 1, wherein the sample isdigested with at least one enzyme, wherein the at least one enzyme istrypsin, chymotrypsin, glutamyl endopeptidase, lysyl endopeptidase,Asp-N, Arg-C, Glu-C, cyanogen bromide (CnBr), or combinations thereof.7. The method of claim 1, wherein the mass spectrometry is liquidchromatography-tandem mass spectrometry analysis (LC-MS/MS).
 8. Themethod of claim 1, wherein: (a) the therapeutic polypeptide comprises aCH1 domain, and wherein the CH1 domain comprises the engineeredmutation; (b) the therapeutic polypeptide comprises a CH2 domain, andwherein the CH2 domain comprises the engineered mutation; or (c) thetherapeutic polypeptide comprises a CH3 domain, and wherein the CH3domain comprises the engineered mutation.
 9. The method of claim 8,wherein the therapeutic polypeptide comprises a CH3 domain, wherein theCH3 domain comprises the engineered mutation, and wherein the engineeredmutation in the CH3 domain of the antibody heavy chain constant regionis T366Y, T366W, T366S, L368A, T394W, T394S, F405A, F405W, Y407T, Y407V,or Y407A.
 10. The method of claim 9, wherein the engineered mutation inthe CH3 domain of the antibody heavy chain constant region is Y407V, andwherein the CH3 domain comprises an amino acid sequence set forth in SEQID NO: 6 (DGSFFLVS) and wherein the digestion produces a peptidefragment comprising the amino acid sequence (SEQ ID NO: 7)TTPPVLDSDGSFFLVSK, (SEQ ID NO: 8) DGSFFLVSKLTV, or (SEQ ID NO: 9)GSFFLVSKLTVD.


11. The method of claim 9, wherein the antibody heavy chain constantregion comprises the amino acid sequence set forth in SEQ ID NO: 3 orSEQ ID NO:
 4. 12. The method of claim 8, wherein the engineered mutationin the CH3 domain of the antibody heavy chain constant region is N434S,wherein the sample is digested with Glu-C and trypsin, and wherein thedigestion produces a peptide fragment consisting of the amino acidsequence (SEQ ID NO: 11) ALHSHYTQK.


13. The method of claim 12, wherein the therapeutic polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 2 or SEQ IDNO:
 3. 14. The method of claim 1, wherein the therapeutic polypeptidecomprising the portion of the antibody heavy chain constant region is anantibody, an Fc-fusion protein, or an immunoadhesin.
 15. The method ofclaim 14, wherein the polypeptide comprising the portion of the antibodyheavy chain constant region is an antibody, and wherein the antibody isa chimeric antibody, a humanized antibody, human antibody, amonospecific antibody, a bispecific antibody, a trispecific antibody, ora multispecific antibody.
 16. The method of claim 15, wherein theantibody is a trispecific antibody comprising four polypeptide chainsthat form three antigen binding sites that specifically bind one or moreantigen targets or target proteins, wherein a first polypeptidecomprises a structure represented by the formula:V_(L2)-L₁-V_(L1)-L₂-CL; the second polypeptide chain comprises astructure represented by the formula:V_(H1)-L₃-V_(H2)-L₄-C_(H1)-hinge-C_(H2)-C_(H3); the third polypeptidechain comprises a structure represented by the formula:V_(H3)-C_(H1)-hinge-C_(H2)-C_(H3); the fourth polypeptide chaincomprises a structure represented by the formula: V_(L3)-CL, whereinV_(L1) is a first immunoglobulin light chain variable domain; V_(L2) isa second immunoglobulin light chain variable domain; V_(L3) is a thirdimmunoglobulin light chain variable domain; V_(H1) is a firstimmunoglobulin heavy chain variable domain; V_(H2) is a secondimmunoglobulin heavy chain variable domain; V_(H3) is a thirdimmunoglobulin heavy chain variable domain; C_(L) is an immunoglobulinlight chain constant domain; C_(H1) is an immunoglobulin C_(H1) heavychain constant domain; and L₁, L₂, L₃ and L₄ are amino acid linkers;wherein the first and second polypeptides form a cross-over lightchain-heavy chain pair; and wherein the second polypeptide chain or thethird polypeptide chain comprises the amino acid sequence (SEQ ID NO: 7)TTPPVLDSDGSFFLVSK,  (SEQ ID NO: 8) DGSFFLVSKLTV, or (SEQ ID NO: 9)GSFFLVSKLTVD.


17. The method of 16, wherein the first polypeptide comprises the aminoacid sequence set forth in SEQ ID NO: 12; the second polypeptidecomprises the amino acid sequence set forth in SEQ ID NO: 13; the thirdpolypeptide comprises the amino acid sequence set forth in SEQ ID NO:14, and the fourth polypeptide comprises the amino acid sequence setforth in SEQ ID NO:
 15. 18. The method of claim 15, wherein the antibodyis conjugated to a drug or a label.
 19. The method of claim 18, whereinthe drug is selected from a chemotherapeutic agent, a cytotoxic agent,or a growth-inhibitory agent.
 20. The method of claim 15, wherein theantibody is a human IgG1 or a human IgG4 antibody.
 21. The method ofclaim 1, wherein the method is for use in pharmacokinetic study of thepolypeptide comprising an antibody heavy chain constant region in amouse, a non-human primate, and a human.